Infectious clones

ABSTRACT

The present invention relates to methods of preparing a DNA comprising steps, wherein (a) a DNA comprising a full length copy of the genomic RNA (gRNA) or an RNA virus; or (b) a DNA comprising one or several fragments of a gRNA of an RNA virus, which fragments code for an RNA dependent RNA polymerase and at least one structural or non-structural protein; or (c) a DNA having a homology of at least 60% to the sequences of (a) or (b); is cloned into a bacterial artificial chromosome (BAC). Additionally, DNAs are provided, which comprise sequences derived from the genomic RNA (gRNA) of a coronavirus which sequences have a homology of at least 60% to the natural sequence of the virus and code for an RNA dependent RNA polymerase and at least one structural or no-structural protein, wherein a fragment of said DNA is capable of being transcribed into RNA which RNA can be assembled to a virion. Further, the use of these nucleic acids for preparation of viral RNA or virions as well as pharmaceutical preparations comprising these DNAs, viral RNAs or virions is disclosed.

FIELD OF THE INVENTION

[0001] This invention relates to methods of preparing a DNA or an RNA,nucleic acids obtainable by this method and their use as vaccines andfor gene therapy.

BACKGROUND OF THE INVENTION

[0002] Advances in recombinant DNA technology have led to progress inthe development of gene transfer between organisms. At this time,numerous efforts are being made to produce chemical, pharmaceutical, andbiological products of economic and commercial interest through the useof gene transfer techniques.

[0003] One of the key elements in genetic manipulation of bothprokaryotic and eukaryotic cells is the development of vectors andvector-host systems. In general, a vector is a nucleic acid moleculecapable of replicating or expressing in a host cell. A vector-hostsystem can be defined as a host cell that bears a vector and allows thegenetic information it contains to be replicated and expressed.

[0004] Vectors have been developed from viruses with both DNA and RNAgenomes. Viral vectors derived from DNA viruses that replicate in thenucleus of the host cell have the drawback of being able to integrateinto the genome of said cell, so they are generally not very safe. Incontrast, viral vectors derived from RNA viruses, which replicate in thecytoplasm of the host cell, are safer than those based on DNA viruses,since the replication occurs through RNA outside the nucleus. Thesevectors are thus very unlikely to integrate into the host cell's genome.

[0005] cDNA clones have been obtained from single-chain RNA viruses withpositive-polarity [ssRNA(+)], for example, picornavirus (Racaniello &Baltimore, 1981); bromovirus (Ahlquist et al., 1984); alphavirus, agenus that includes the Sindbis virus; Semliki Forest virus (SFV) andthe Venezuelan equine encephalitis virus (VEE) (Rice et al., 1987;Liljeström and Garoff, 1991; Frolov et al., 1996; Smerdou andLiljestrom, 1999); flavivirus and pestivirus (Rice and Strauss, 1981;Lai et al., 1991; Rice et al., 1989); and viruses of the Astroviridaefamily (Geigen-muller et al., 1997). Likewise, vectors for theexpression of heterologous genes have been developed from clones of DNAcomplementary to the genome of ssRNA(+) virus, for example alphavirus,including the Sindbis virus, Semliki Forest virus (SFV), and theVenezuelan equine encephalitis (VEE) virus (Frolov et al., 1996;Liljeström, 1994; Pushko et al., 1997). However, all methods ofpreparing recombinant viruses starting from RNA viruses are stillcomplicated by the fact that most of the viruses comprise sequenceswhich are toxic for bacteria. Preparing a cDNA of the viral RNA andsubcloning of the cDNA in bacteria therefore often leads to deletion orrearangement of the DNA sequences in the bacterial host. For thispurpose most of the commonly used subcloning and expression vectorscannot be used for preparation of large DNA sections derived fromrecombinant RNA viruses. However, obtaining vectors, which can carrylong foreign DNA sequences is required for a number of aspects in thedevelopment of pharmaceuticals, specifically vaccines.

[0006] The coronaviruses are ssRNA(+) viruses that present the largestknown genome for an RNA virus, with a length comprised between about 25and 31 kilobases (kb) (Siddell, 1995; Lai & Cavanagh, 1997; Enjuanes etal., 1998). During infection by coronavirus, the genomic RNA (gRNA)replicates and a set of subgenomic RNAs (sgRNA) of positive and negativepolarity is synthesized (Sethna et al., 1989; Sawicki and Sawicki, 1990;van der Most & Spaan, 1995). The synthesis of the sgRNAs is anRNA-dependent process that occurs in the cytoplasm of the infected cell,although its precise mechanism is still not exactly known.

[0007] The construction of cDNAs that code defective interfering (DI)genomes (deletion mutants that require the presence of a complementingvirus for their replication and transcription) of some coronaviruses,such as the murine hepatitis virus (MHV), infectious bronchitis virus(IBV), bovine coronavirus (BCV) (Chang et al., 1994), and porcinegastroenteritis virus (TGEV) (Spanish Patent Application P9600620;Mendez et al., 1996; Izeta et al., 1999; Sánchez et al., 1999) has beendescribed. However, the construction of a cDNA clone that codes acomplete genome of a coronavirus has not been possible due to the largesize of and the toxic sequences within the coronavirus genome.

[0008] In summary, although a large number of viral vectors have beendeveloped to replicate and express heterologous nucleic acids in hostcells, the majority of the known vectors for expression of heterologousgenes are not well suited for subcloning of RNA viruses. Further, theviral vectors so obtained present drawbacks due to lack of speciesspecificity and target organ or tissue limitation and to their limitedcapacity for cloning, which restricts the possibilities of use in bothbasic and applied research.

[0009] Hence there is a need for methods to develop new vectors forexpression of heterologous genes that can overcome the aforesaidproblems. In particular, it would be advantagous to have large vectorsfor expression of heterologous genes with a high level of safety andcloning capacity, which can be designed so that their speciesspecificity and tropism can be controlled.

SUMMARY OF THE INVENTION

[0010] According to the present invention the above problems are solvedby a method of preparing a DNA comprising steps, wherein

[0011] (a) a DNA comprising a full length copy of the genomic RNA (gRNA)of an RNA virus; or

[0012] (b) a DNA comprising one or several fragments of a gRNA of an RNAvirus, which fragments code for an RNA dependent RNA polymerase and atleast one structural or non-structural protein; or

[0013] (c) a DNA having a homology of at least 60% to the sequences of(a) or (b); is cloned into a bacterial artificial chromosome (BAC).

[0014] Surprisingly, the present inventors found that the problemsencountered by the prior art methods to subclone and express large DNAsequences derived from viral gRNA can be overcome by using BACs as acloning vector. The use of BACs has the particular advantage that thesevectors are present in bacteria in a number of one or two copies percell, which considerably limits the toxicity and reduces thepossibilities of interplasmid recombinantion.

[0015] The invention further provides methods of preparing a viral RNAor a virion comprising steps, wherein a DNA is prepared according to oneof the above methods, the DNA is expressed and the viral RNA or thevirion is isolated. Further, methods of preparing pharmaceuticals,specifically vaccines comprising the steps of the above methods toprepare a DNA are disclosed.

[0016] According to another aspect of the present invention provides aDNA comprising sequences derived from the genomic RNA (gRNA) of acoronavirus which sequences have a homology of at least 60% to thenatural sequence of the coronavirus and code for an RNA dependent RNApolymerase and at least one structural or non-structural protein,wherein a fragment of said DNA is capable of being transcribed into RNAand which RNA can be assembled to a virion. The present invention alsoencompasses methods of preparing respective DNAs.

[0017] The present invention further provides vectors, more specificallybacterial artificial chromosomes (BACs) comprising respective nucleicacids. According to a further embodiment the present invention isdirected to host cells and infectious, attenuated or inactivated virusescomprising the DNAs or RNAs of the present invention.

[0018] The invention also provides pharmaceutical preparations, such asmono- or multivalent vaccines comprising nucleic acids, vectors, hostcells or virions of the present invention.

[0019] Finally, the present invention provides methods for producing avirion or a viral RNA comprising steps, wherein a DNA according to thepresent invention is transcribed and the virions or viral RNAs arerecovered, as well as viral RNAs obtainable by this method.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows the construction of a cDNA clone that codes aninfective RNA of TGEV.

[0021]FIG. 1A shows the genetic structure of the TGEV, with the names ofthe genes indicated by letters and numbers (1a, 1b, S, 3a, 3b, E, M, N,and 7).

[0022]FIG. 1B shows the cDNA-cloning strategy, which consisted incompleting the DI-C genome. Deletions Δ1, Δ2, and Δ3 that have beencompleted to reestablish the full length of the cDNA are indicated. Thenumbers located beneath the structure of the DI-C genome indicate thenucleotides that flank each deletion in said DI-C genome.

[0023]FIG. 1C shows the four cDNA fragments constructed to completedeletion Δ1 and the position of the principal restriction sites usedduring joining. The insertion of fragment Δ1 produced an increase in thetoxicity of the cDNA.

[0024]FIG. 2 shows the structure of the pBeloBAC plasmid (Wang et al.,1997) used in cloning the infective cDNA of TGEV. The pBeloBAC plasmidwas provided by H. Shizuya and M. Simon (California Institute ofTechnology) and includes 7,507 base pairs (bp) that contain thereplication origin of the F factor of E. coli (oriS), the genesnecessary to keep one single copy of the plasmid per cell (parA, parB,parC, and repE), and the chloramphenicol-resistance gene (CM^(r)). Thepositions of the T7 and SP6 promoters and of the unique restrictionsites are indicated. CosN: site cosN of lambda to facilitate theconstruction of the pBAC plasmid; lac Z: β-galactosidase gene. SequenceloxP used during the generation of the plasmid is also indicated.

[0025]FIG. 3 shows the structure of the basic plasmids used in theconstruction of TGEV cDNA. The pBAC-TcDNA^(ΔClaI) plasmid contains allthe information of the TGEV RNA except for one ClaI-ClaI fragment of5,198 bp. The cDNA was cloned under the immediately early (IE) promoterof expression of cytomegalovirus (CMV) and is flanked at the 3′-end by apoly(A) tail with 24 residues of A, the ribozyme of the hepatitis deltavirus (HDV), and the termination and polyadenylation sequences of bovinegrowth hormone (BGH). The pBAC-B+C+D5′ plasmid contains the ClaI-ClaIfragment required to complete the pBAC-TcDNA^(ΔClaI) until the cDNA isfull length. The pBAC-TcDNA^(FL) plasmid contains the full-length cDNAof TGEV. SAP: shrimp alkaline phosphatase.

[0026]FIG. 4 shows the differences in the nucleotide sequence of the Sgene of the clones of TGEV PUR46-MAD (MAD) and C11. The numbers indicatethe positions of the substituted nucleotides, considering as nucleotideone of each gene the A of the initiating codon. The letters within thebars indicate the corresponding nucleotide in the position indicated.The letters located beneath the bars indicate the amino acid (aa)substitutions coded by the nucleotides that are around the indicatedposition. Δ6 nt indicates a 6-nucleotide deletion. The arrow indicatesthe position of the termination codon of the S gene.

[0027]FIG. 5 shows the strategy followed to rescue the infective TGEVfrom the full-length TGEV cDNA. The pBAC-TcDNA^(FL) plasmid wastransfected to ST cells (pig testicle cells), and 48 h aftertransfection, the supernatant was used to infect new ST cells. The viruswas passed at the times indicated. At each passage, aliquots ofsupernatant and of cellular monolayer were collected for virus titrationand isolation of RNA for RT-PCR analysis, respectively. vgRNA:full-length viral RNA.

[0028]FIG. 6 shows the cytopathic effect (CPE) produced by the TGEV cDNAin the transfected ST cells. The absence of CPE in non-transfected(control) ST cells (FIG. 6A) and the CPE observed 14 and 20 h aftertransfection with pBAC-TcDNA^(FL) in ST cells are shown (FIGS. 6B and6C, respectively).

[0029]FIG. 7 shows the evolution of the viral titer with the passage. Agraph representing the viral titer in the supernatant of two series ofcellular monolayers (1 and 2) at different passages after transfectionwith pBAC-TcDNA^(FL) is shown. Mock 1 and-2 refer to nontransfected STcells. TcDNA 1 and 2 refer to ST cells transfected with pBAC-TcDNA^(FL).

[0030]FIG. 8 shows the results of the analysis of the sequence of thevirus recovered after transfecting ST cells with pBAC-TcDNA^(FL). Thestructure of the TGEV genome is indicated at the top of the figure.Likewise, the differences in the sequence of nucleotides (geneticmarkers) between the virus recovered from the pBAC-TcDNA (TcDNA)plasmid, and TGEV clones C8 and C11 are indicated. The positions of thedifferences between the nucleotides are indicated by the numbers locatedover the bar. The cDNA sequences of the TcDNA virus and of clone C11were determined by sequencing the fragments obtained by RT-PCR(reverse-transcription and polymerase chain reaction). The sequence ofclone C8 is being sent for publication (Penzes et al., 1999) and isincluded at the end of this patent. The restriction patterns are shownwith ClaI and DraIII of the fragments obtained by RT-PCR that includenucleotides 18,997 and 20,990 of the TcDNA and C8 viruses. Therestriction patterns show the presence or absence of ClaI and DraIIIsites in the cDNA of these viruses. The result of this analysisindicated that the TcDNA virus recovered had the S-gene sequenceexpected for isolate C11. MWM: molecular weight markers.

[0031]FIG. 9 shows the results of the RT-PCR analysis of the virusrecovered. The viral RNA was expressed under the control of the CMVpromoter recognized by the cellular polymerase pol II. In principle,this RNA could undergo splicing during its transport to the cytoplasm.To study whether this was the case, the sites of the RNA with a highprobability of splicing were determined using a program for predictingsplicing sites in sequences of human DNA (Version 2.1.5.94, Departmentof Cell Biology, Baylor College of Medicine) (Solovyev et al., 1994).The potential splicing site with maximum probability of cut had thedonor site at nt 7,243 and the receiver at nt 7,570 (FIG. 9A). To studywhether this domain had undergone splicing, a RT-PCR fragment flanked bynt 7,078 and nt 7,802 (FIG. 9B) was prepared from RNA of passages 0 and2 of nontransfected cultures (control), or from ST cells transfectedwith TcDNA with the ClaI fragment in reverse orientation(TcDNA^(FL(−ΔClaI)RS)), or in the correct orientation (TcDNA^(FL)), andthe products resulting from the RT-PCR were analyzed in agarose gels.The results obtained are shown in FIGS. 9C (passage 0) and 9D (passage2).

[0032]FIG. 10 shows the results of the immunofluorescence analysis ofthe virus produced in cultures of ST cells transfected with TcDNA.Staining for immunofluorescence was done with antibodies specific forthe TGEV PUR46-MAD isolate, and for the virus recovered aftertransfection with the pBAC-TcDNA plasmid. For this, TGEV-specificmonoclonal antibodies were used which bind to both isolates or only toPUR46-MAD (Sanchez et al., 1990). The result confirmed that the TcDNAvirus had the expected antigenicity. The specific polyclonal antiserumfor TGEV bound to both viruses, but not to the uninfected cultures, andonly the expected monoclonal antibodies specific for the S (ID.B12 and6A.C3), M (3B.B3), and N (3B.D8) proteins bound to the TcDNA virus(Sánchez et al., 1999).

[0033]FIG. 11 shows the expression of GUS under differenttranscription-regulatory sequences (TRSs) that vary flanking region 5′of the intergenic (IG) sequence. Minigenome M39 was cloned under thecontrol of the CMV promoter. Inserted into this minigenome was amultiple cloning sequence (PL1, 5′-CCTAGGATTTAAATCCTAAGG-3′; SEQ ID NO:2) and the transcription unit formed by the selectedtranscription-regulating sequences (TRS), another multiple cloningsequence (PL2, 5′-GCGGCCGCGCCGGCGAGGCCTGTCGAC-3′; SEQ ID NO: 3; or PL3,5′-GTCGAC-3′; SEQ ID NO: 4), sequences with the structure of a Kozak(Kz) domain, the β-glucuronidase (GUS) gene, and another multiplecloning site (PL4, 5′-GCTAGCCCAGGCGCGCGGTACC-3′; SEQ ID NO: 5). Thesesequences ¹were flanked at the 3′-end by the 3′-sequence of minigenomeM39, the HDV ribozyme, and the termination and polyadenylation sequencesof BGH. The TRSs had a different number (0, −3, −8, and −88) ofnucleotides at the 5′-end of the IG sequence (CUAAAC)¹, and came fromthe N, S, or M genes, as indicated. ST cells were transfected with thedifferent plasmids, were infected with the complementing virus(PUR46-MAD), and the supernatants were passed 6 times. The GUS activityin the infected cells was determined by means of the protocol describedby Izeta (Izeta et al., 1999). The results obtained by relating the GUSactivity to the passage number are collected in FIG. 11B.

[0034]FIG. 12 shows the expression of GUS under different TRSs that varyin the 3′-flanking region of the IG sequence (see FIG. 11A). Using thistranscription unit with the 5′-flanking region corresponding to the −88nt of the N gene of TGEV plus the IG sequence (CUAAAC), the 3′-flankingsequences were modified. These sequences corresponded to those of thedifferent TGEV genes (S, 3a, 3b, E, M, N, and 7), as is indicated inFIG. 12A. In two cases, 3′-sequences were replaced by others thatcontained a restriction site (SalI) and an optimized Kozak sequence(Kz), or by a sequence identical to the one that follows the first IGsequence located following the leader of the viral genome. The activityof GUS in the infected cells was determined by means of the protocoldescribed above (Izeta et al., 1999). cL12 indicates a sequence of 12nucleotides identical to that of 3′-end of the ‘leader’ sequence of theTGEV genome (see the virus sequence indicated at the end). The resultsobtained by relating the expression of GUS to the passage number arecollected in FIG. 12B.

[0035]FIG. 13 shows the effect of the site of insertion of the module ofexpression in the minigenome over the levels of GUS expression. The GUStranscription unit, containing −88 nt of the N gene flanking the 5′-endof the IG sequence (CUAAAC), and the Kz sequences flanking the 3′-end(see FIG. 12A), was inserted into four single restriction sites inminigenome M39 (FIG. 13A) to determine if all these sites were equallypermissive for the expression of the heterologous gene. ST cells weretransfected with these plasmids and infected with the complementingvirus (PUR46-MAD). The GUS activity in the infected cells was determinedat passage 0 (P0) following the protocol described above (Izeta et al.,1999). The results obtained are collected in FIG. 13B.

DETAILED DESCRIPTION OF THE INVENTION

[0036] According to the present invention methods of preparing a DNA areprovided, which comprise steps, wherein

[0037] (a) a DNA comprising a full length copy of the genomic RNA (gRNA)of an RNA virus; or

[0038] (b) a DNA comprising one or several fragments of a gRNA of an RNAvirus, which fragments code for an RNA dependent RNA polymerase and atleast one structural or non-structural protein; or

[0039] (c) a DNA having a homology of at least 60% to the sequences of(a) or (b);

[0040] is cloned into a bacterial artificial chromosome (BAC).

[0041] According to the present application a “bacterial artificialchromosome” is a DNA sequence which comprises the sequence of the Ffactor. Plasmids containing this sequences, so-called F plasmids, arecapable of stably maintaining heterologous sequences longer than 300 Kbin low copy number (one or two copies per cell). Respective BACs areknown in the art (Shizuya et al., 1992).

[0042] According to the present invention the DNA cloned into the BAChas a homology of at least 60%, preferably 75% and more preferably 85 or95%, to a natural sequence of an RNA virus. Sequence homology ispreferably determined using the Clustal computer program available fromthe European Bioinformatics Institute (EBI).

[0043] According to the methods of the present invention the DNA clonedinto the BAC may further comprise sequences coding for several or allexcept one of the structural or non-structural proteins of the virus.

[0044] In a preferred embodiment of the present invention the DNA clonedinto the BAC further comprises sequences encoding one or severalheterologous gene. According to the present application a gene ischaracterized as a “heterologous gene” if it is not derived from thevirus which was used as a source for the genes encoding the RNAdependent RNA polymerase and the structural or non-structural protein. A“heterologous gene” thus also refers to genes derived from one type ofvirus and expressed in a vector comprising sequences derived fromanother type of virus. Any heterologous gene of interest can be insertedinto the nucleic acids of the present invention. The insertion of genesencoding one or several peptides or proteins which are recognised as anantigen from an infectious agent by the immune system of a mammal isespecially preferred. Alternatively, the method of the present inventionmay be performed using heterologous genes encoding at least one moleculeinterfering with the replication of an infectious agent or an antibodyproviding protection against an infectious agent. The heterologoussequences may contain sequences encoding an immune modulator, acytokine, an immonenhancer and/or an anti-inflammatory compound.

[0045] The method of the present invention may be performed using a DNAfor cloning into a BAC that has any size. However, specific advantagesover the known methods to prepare subcloned DNA from viral are obtained,if large sequences are used. The DNA cloned into the BAC may thuscomprise a length of at least 5 Kb, wherein DNA with a size of at least15, 25 or 30 Kb is specifically preferred.

[0046] According to specifically preferred embodiments of the presentinvention methods are provided, wherein the BAC comprises a sequencecontrolling the transcription of the DNA cloned into the BAC. This willallow transcription of the viral RNA and thus enable expression of thevirus. Any sequence controlling transcription known in the art may beused for this purpose, including sequences driving the expression ofgenes derived from DNA or RNA genomes. The use of the immediately early(IE) promoter of cytomegalovirus (CMV) is preferred.

[0047] The DNA cloned into the BAC may also be flanked at the 3′-end bya poly(A)tail. The nucleic acid may comprise termination and/orpolyadenylation sequences of bovine growth hormone (BGH). Additionallyor alternatively, the nucleic acids may comprise sequences encoding aribozyme, for example the ribozyme of the hepatitis δ virus (HDV).

[0048] Additional advantages may be achieved if at least one of thegenes of the virus has been modified by substituting, deleting or addingnucleotides. For example the gene controlling tropism of the virus maybe modified to obtain viruses with altered tropism. Alternativly, thegene controlling tropism of the virus has been substituted with therespective gene of another virus. The modification is preferablyperformed in the S, M and/or N genes of the virus.

[0049] In a preferred embodiment of the present invention a method isprovided, wherein the DNA cloned into the BAC is capable of beingtranscribed into RNA which RNA can be assembled to an virion. The virionmay be an infectious, attenuated, replication defective or inactivatedvirus.

[0050] Any RNA virus may be used in the methods of the invention. Thevirus can for example be a picornavirus, flavivirus, togavirus,coronavirus, toroviruses, arterivurses, calcivirus, rhabdovirus,paramixovirus, filovirus, bornavirus, orthomyxovirus, bunyavirus,arenavirus or reovirus. The use of viruses naturally having a plusstrand genome is preferred.

[0051] Additionally, the present invention provides methods of preparinga viral RNA or a virion comprising steps, wherein a DNA is preparedaccording to one of above methods, the DNA is expressed in a suitablehost cell and the viral RNA or the virion is isolated from that hostcell. Any of methods for isolating viruses from the cell culture knownin the art may be used. Alternatively, methods of preparing a viral RNAor a virion are disclosed, wherein the DNA of the present invention istranscribed or translated using chemicals, biological reagents and/orcell extracts and the viral RNA or the virion is subsequently isolated.For certain embodiments, the virus may subsequently be inactivated orkilled.

[0052] The invention also provides methods for preparing apharmaceutical composition comprising steps, wherein a DNA, a viral RNAor a virion is prepared according to one of the above methods and issubsequently mixed with a pharmaceutically acceptable adjuvans and/orcarrier. A large number of adjuvans and carriers and diluents are knownin the prior art and may be used in accordance with the presentinvention. The pharmaceutical is preferably a vaccine for protectinghumans or animals against an infectious disease. The pharmaceutical canadvantageously also be used for gene therapy.

[0053] The present invention further provides for the first time a DNAcomprising sequences derived from the genomic RNA (gRNA) of acoronavirus which sequences have a homology of at least 60% to thenatural sequence of the coronavirus and code for an RNA dependent RNApolymerase and at least one structural or non-structural protein,wherein a fragment of said DNA is capable of being transcribed into RNAwhich can be assembled to a virion.

[0054] According to the present invention the term “sequence derivedfrom a coronavirus” is used to refer to a nucleic acid sequence whichhas a homology of at least 60%, preferably 75% and more preferably 85 or95%, to a natural sequence of a coronavirus. Sequence homology can bedetermined using the Clustal computer program available from theEuropean Bioinformatics Institute (EBI).

[0055] The term “coronavirus” is used according to the present inventionto refer to a group of viruses having a single molecule of linear,positive sense, ssRNA of 25 to 33 Kb. These viruses usually contain 7 to10 structural genes, i.e. genes encoding proteins that determine theviral structure. These genes are typically arranged in the viral genomein the order of 5′replicase-(hemagglutinin-esterase)-spike-envelope-membrane-nucleoprotein-3′.Additionally the viral genome may comprise a number of non-structuralgenes which encode a nested set of mRNAs with a common 3′ end and arelargely non-essential.

[0056] The term “capable of being transcribed into RNA which can beassembled into a virion” is used to characterize a DNA sequence,which—once introduced into a suitable host cell—will be transcribed intoRNA and generate virions. The virions are preferably infectious viruses,but may also be inactivated, attenuated or replication defective virusescomprising said RNA. Preferably all the information necessary forexpression of the virion is encoded by the DNA sequence of the presentinvention.

[0057] The nucleic acids of the present invention may further comprise asequence encoding one or several heterologous genes of interest.According to the present invention a gene is characterized as a“heterologous gene” if it is not derived from the coronavirus which wasused as a source for the genes encoding the RNA dependent RNA polymeraseand the structural or non-structural protein. A “heterologous gene” thusalso refers to genes derived from one type of coronavirus and expressedin a vector comprising sequences derived from another type ofcoronavirus. Any heterologous gene of interest can be inserted into thenucleic acids of the present invention. The insertion of genes encodingpeptides or proteins which are recognised as an antigen from an-infectious agent by the immune system of a mammal is especiallypreferred. The heterologous gene may thus encode at least one antigensuitable for inducing an immune response against an infectious agent, atleast one molecule interfering with the replication of an infectiousagent or an antibody providing protection against an infectious agent.Alternatively or additionally, the heterologous gene may encode animmune modulator, a cytokine, an immonenhancer or an anti-inflammatorycompound.

[0058] The fragment of the DNA according to the present invention whichis transcribed into RNA preferably has a size of at least 25 Kb.Fragments with a size of at least 30 Kb are especially preferred.

[0059] According to a preferred embodiment of the present invention theDNA further comprises sequences derived from a coronavirus coding forseveral or all except one of the structural or nonstructural proteins ofa coronavirus. Alternatively, the DNA of the present invention furthercomprises sequences coding for all of the structural or non-structuralproteins of a coronavirus.

[0060] According to a further embodiment, the nucleic acids of thepresent invention comprise a sequence controlling the transcription of asequence derived from a coronavirus gRNA. Any sequence controllingtranscription known in the art may be used for this purpose, includingsequences driving the expression of genes derived from DNA or RNAgenomes. The use of the immediately early (IE) promoter ofcytomegalovirus (CMV) is preferred.

[0061] The nucleic acid according to the present invention may also beflanked at the 3′-end by a poly(A)tail. The nucleic acid may comprisetermination and/or polyadenylation sequences of bovine growth hormone(BGH). Additionally or alternatively, the nucleic acids may comprisesequences encoding a ribozyme, for example the ribozyme of the hepatitisδ virus (HDV).

[0062] The nucleic acids of the present invention may comprise sequencesderived from any coronavirus, for example derived from an isolate of theporcine transmissible gastroenteritis virus (TGEV), murine hepatititsvirus (MHV), infectious bronchitis virus (IBV), bovine coronavirus(BoCV), canine coronavirus (CCoV), feline coronavirus (FcoV) or humancoronavirus. According to a preferred embodiment the nucleic acid isderived from a transmissable gastroenteritis virus.

[0063] According to a further embodiment of the present invention, theDNAs of the present invention are part of a plasmid, preferably part ofa bacterial artificial chromosome (BAC).

[0064] The present invention further provides host cells comprisingrespective nucleic acids or vectors. The host cells may be eucaryotes orprocaryotes. Alternatively, the present invention provides virionscomprising a nucleic acid according the present invention. Respectivevirions may for example be isolated from cell cultures transfected orinfected with the nucleic acids of the present invention.

[0065] According to a further embodiment, the present invention providesmethods for producing a virion or a viral RNA comprising steps, whereina DNA of the present invention is introduced into a host cell, hostcells containing the DNA are cultivated under conditions allowing theexpression thereof and the virion or viral RNA is recovered.Additionally, methods for producing a virion or a viral RNA areprovided, wherein a DNA of the present invention is mixed in vitro withchemicals, biological reagents and/or cell extracts under conditionsallowing the expression of the DNA and the virion or viral RNA isrecovered. The present invention also encompasses the virions and viralRNAs obtainable by either of the above methods. RNAs and virionscarrying a heterologous gene are preferred. The viruses so obtained mayhave the form of an infectious, attenuated, replication defective orinactivated virus.

[0066] The virus may comprise modified genes, for example a modified S,N or M gene. In a specific embodiment of the present invention themodification of the S, N or M gene gives raise to an attenuated virus ora virus with altered tropism.

[0067] According to a further embodiment the invention provides apharmaceutical preparation comprising nucleic acids, host cells orvirions according to the present invention. According to a preferredembodiment the pharmaceutical preparation is a vaccine capable ofprotecting an animal against deseases caused by an infectious agent. Thevaccine may for example comprise sequences of at least one antigensuitable for inducing an immune response against the infectious agent oran antibody providing protection against said infectious agent. Thevaccine may comprise a DNA expressing at least one molecule interferingwith the replication of the infectious agent. Alternatively the vaccinemay comprise a vector expressing at least one antigen capable ofinducing a systemic immune response and/or an immune response in mucousmembranes against different infectious agents that propagate inrespiratory, intestinal mucous membranes or in other tissues. Thevaccine may also be a multivalent vaccine capable of protecting ananimal against the infection caused by more than one infectious agent,that comprises more than one nucleic acid of the present invention eachof which expresses an antigen *;0 capable of inducing an immune responseagainst each of said infectious agents, or antibodies that provideprotection against each one of said infectious agents or other moleculesthat interfere with the replication of any infectious agent.

[0068] The vaccines of the present invention may further comprise any ofthe pharmaceutically acceptable carriers or diluents known in the stateof the art.

[0069] The present invention further provides methods for preparing aDNA of the present invention comprising steps, wherein an interferingdefective genome derived from a coronavirus is cloned under theexpression of a promotor into a BAC vector and the deletions within thedefective genome are re-inserted. The method may further comprise steps,wherein toxic sequences within the viral genome are identified beforere-insertion into the remaining genomic DNA. Preferably, the toxicsequences within the viral genome are the last sequences to bere-inserted before completing the genome. According to the presentinvention this method is suitable to yield infectious clones ofcoronaviruses which are stable in bacteria for at least 80 generationsand thus provides a very efficient cloning vector.

[0070] The present invention provides the development of infectiveclones of cDNA derived from coronavirus (Almazan et al., 2000), as wellas vectors constructed from said infective clones that also includeheterologous nucleic acid sequences inserted into said clones. Theinfective clones and vectors provided by this invention have numerousapplications in both basic and applied research, as well as a highcloning capacity, and can be designed in such a way that their speciesspecificity and tropism can be easily controlled.

[0071] This patent describes the development of a method that makes itpossible to obtain, for the first time in the history of coronavirus, afull-length infective cDNA clone that codes the genome of a coronavirus(Almazan et al., 2000).

[0072] A new vector or system of expression of heterologous nucleicacids based on a coronavirus generated from an infective cDNA clone thatcodes the genomic RNA (gRNA) of a coronavirus has been developed. In oneparticular realization of this invention, the coronavirus is the porcinetransmissible gastroenteritis virus (TGEV).

[0073] The new system of expression can be used in basic or appliedresearch, for example, to obtain products of interest (proteins,enzymes, antibodies, etc.), as a vaccinal vector, or in gene therapy inboth humans and animals. The infective coronavirus obtained from theinfective cDNA clone can be manipulated by conventional geneticengineering techniques so that new genes can be introduced into thegenome of the coronavirus, and so that these genes can be expressed in atissue- and species-specific manner to induce an immune response or forgene therapy. In addition, the expression has been optimized by theselection of new transcription-regulating sequences (TRS) that make itpossible to increase the levels of expression more than a hundredfold.

[0074] The vectors derived from coronavirus, particularly TGEV, presentseveral advantages for the induction of immunity in mucous membraneswith respect to other systems of expression that do not replicate inthem: (i) TGEV infects intestinal and respiratory mucous membranes(Enjuanes and Van der Zeijst, 1995), that is, the best sites forinduction of secretory immunity; (ii) its tropism can be controlled bymodifying the S (spike) gene (Ballesteros et al., 1997); (iii) there arenonpathogenic strains for the development of systems of expression thatdepend on complementing virus (Sánchez et al., 1992); and (iv)coronaviruses are cytoplasmic RNA viruses that replicate without passingthrough an intermediate DNA stage (Lai and Cavanagh, 1997), making itsintegration into the cellular chromosome practically impossible.

[0075] The procedure that has made it possible to recover an infectivecoronavirus from a cDNA that codes the gRNA of a coronavirus includesthe following strategies:

[0076] (i) expression of the RNA of the coronavirus under the control ofan appropriate promoter;

[0077] (ii) cloning of the genome of the coronavirus in bacterialartificial chromosomes (BACs);

[0078] (iii) identification of the sequences of cDNA of the coronavirusthat are directly or indirectly toxic to bacteria;

[0079] (iv) identification of the precise order of joining of thecomponents of the cDNA that codes an infective RNA of coronavirus(promoters, transcription-termination sequences, polyadenylationsequences, ribozymes, etc.); and

[0080] (V) identification of a group of technologies and processes(conditions for the growth of BACs, modifications to the purificationprocess of BAC DNA, transformation techniques, etc.) that, incombination, allow the efficient rescue of an infective coronavirus of acDNA.

[0081] The promoter plays an important role in increasing the expressionof viral RNA in the nucleus, where it is synthesized, to be transportedto the cytoplasm later on.

[0082] The use of BACs constitutes one of the key points of theprocedure of the invention. As is known, cloning of eukaryotic sequencesin bacterial plasmids is often impossible due to the toxicity of theexogenous sequences for bacteria. In these cases, the bacteria usuallyeliminate toxicity by modifying the introduced sequences. Nevertheless,in the strategy followed in this case, to avoid the possible toxicity ofthese viral sequences, the necessary clonings were carried out to obtaina complete cDNA of the coronavirus in BACs. These plasmids appear inonly one copy or a maximum of two per cell, considerably limiting theirtoxicity and reducing the possibilities of interplasmid recombination.

[0083] Through the identification of the bacteriotoxic cDNA sequences ofthe coronavirus, the construction of the cDNA that codes the completegenome of a coronavirus can be completed, with the exception of thetoxic sequences, which are added in the last step of construction of thecomplete genome, that is, just before transfection in eukaryotic cells,avoiding their modification by the bacteria.

[0084] One object of the present invention therefore consists in aninfective double-chain cDNA clone that codes the gRNA of a coronavirus,as well as the procedure for obtaining it.

[0085] An additional object of this invention consists in a set ofrecombinant viral vectors that comprises said infective clone andsequences of heterologous nucleic acids inserted into said infectiveclone.

[0086] An additional object of this invention consists in a method forproducing a recombinant coronavirus that comprises the introduction ofsaid infective clone into a host cell, the culture of the transformedcell in conditions that allow the replication of the infective clone andproduction of the recombinant coronavirus, and recovering therecombinant coronavirus from the culture.

[0087] Another object of this invention consists in a method forproducing a modified recombinant coronavirus that comprises introducingthe recombinant viral vector into a host cell, cultivating it inconditions that allow the viral vector to replicate and the modifiedrecombinant coronavirus to be produced, and recovering the modifiedrecombinant coronavirus from the culture. Another object of thisinvention consists in a method for producing a product of interest thatcomprises cultivating a host cell that contains said recombinant viralvector in conditions that allow the expression of the sequence ofheterologous DNA.

[0088] Cells containing the aforementioned infective clones orrecombinant viral vector constitute another object of the presentinvention.

[0089] Another object of this invention consists in a set of vaccinesthat protect animals against infections caused by infectious agents.These vaccines comprise infective vectors that express at least oneantigen adequate for inducing an immune response against each infectiveagent, or at least one antibody that provides protection against saidinfective agent, along with a pharmaceutically acceptable excipient. Thevaccines can be mono- or multivalent, depending on whether the vectorsexpress one or more antigens capable of inducing an immune response toone or more infectious agents, or, alternatively, one or more antibodiesthat provide protection against one or more infectious agents.

[0090] Another object provided by this invention comprises a method ofanimal immunization that consists in the administration of said vaccine.

[0091] The invention provides a cDNA clone that codes the infective RNAof a coronavirus, henceforth the infective clone of the invention, whichcomprises: (1) a copy of the complementary DNA (cDNA) to the infectivegenomic RNA (gRNA) of a coronavirus or the viral RNA itself; and (2) anexpression module for an additional gene, which includes optimizedtranscription-promoting sequences.

[0092] In one particular realization of this invention, the coronavirusis a TGEV isolate, in particular, the PUR46-MAD isolate (Sánchez et al.,1990), modified by the replacement of the S gene of this virus by the Sgene of the clone C11 TGEV isolate or the S-gene of a canine or humancoronavirus.

[0093] The transcription-promoting sequence, or promoter, is an RNAsequence located at the 5′-terminal end of each messenger RNA (mRNA) ofcoronavirus, to which the viral polymerase RNA binds to begin thetranscription of the messenger RNA (mRNA). In a particular and preferredembodiment the viral genome is expressed from a cDNA using the IEpromoter of CMV, due to the high level of expression obtained using thispromoter (Dubensky et al., 1996), and to previous results obtained inour laboratory that indicated that large defective genomes (9.7 kb and15 kb) derived from the TGEV coronavirus expressed RNAs that did notundergo splicing during their transport from the nucleus, where they aresynthesized, to the cytoplasm.

[0094] The infective clone of the invention also contains atranscription termination sequence and a polyadenylation signal such asthat coming from the BGH gene. These termination sequences have to beplaced at the 3′-end of the poly(A) tail. In one particular realization,the infective clone of the invention contains a poly(A) tail of 24residues of A and the termination and polyadenylation sequences of theBGH separated from the poly(A) tail by the sequence of the HDV ribozyme.

[0095] The plasmid into which the infective cDNA of the virus has beeninserted is a DNA molecule that possesses a replication origin, and istherefore potentially capable of replicating in a suitable cell. Theplasmid used is a replicon adequate for maintaining and amplifying theinfective clone of the invention in an adequate host cell such as abacterium, for example, Escherichia coli. The replicon generally carriesa gene of resistance to antibiotics that allows the selection of thecells that carry it (for example, cat).

[0096] In Example 1, the construction of an infective clone of TGEVunder the control of the IE promoter of CMV is described. The 3′-end ofthe cDNA appears flanked by a 24 nt poly(A) sequence, the HDV ribozyme,and the transcription termination sequence of the BGH.

[0097] The procedure for obtaining the infective clone of the inventioncomprises constructing the full-length cDNA from the gRNA of acoronavirus and joining the transcription-regulating elements.

[0098] The cDNA that codes the infective gRNA of a coronavirus wasobtained from a DI genome derived from a coronavirus cloned as a cDNAunder the control of an appropriate promoter in a BAC, for the purposeof increasing the cDNA's stability. Then the bacteriotoxic sequenceswere identified and, for the purpose of eliminating that toxicity, saidtoxic sequences were removed and inserted at the end of the constructionof the complete genome, just before transfection in eukaryotic cells.The viral progeny can be reconstituted by means of transfection of theBAC plasmid that contains the coronavirus genome in eukaryotic cellsthat support viral replication.

[0099] The transcription-regulating elements are joined by means ofconventional techniques (Maniatis et al., 1989).

[0100] The infective clone of the invention can be manipulated byconventional genetic engineering techniques to insert at least onesequence of a heterologous nucleic acid that codes a determinedactivity, under the control of the promoter that is present in theinfective clone and of the regulating sequences contained in theresulting expression vector.

[0101] The infective clone of the invention presents numerousapplications; for example, it can be used both in basic research, forexample, to study the mechanism of replication and transcription ofcoronaviruses, and in applied research, for example, in the developmentof efficient systems of expression of products of interest (proteins,enzymes, antibodies, etc.).

[0102] Appropriate cells can be transformed from the infective cDNAclone of the invention, and the virions obtained containing the completegenome of the coronavirus can be recovered. Therefore, the inventionmoreover provides a method for producing a recombinant coronavirus thatcomprises the introduction of an infective cDNA of the invention into ahost cell, the culture of said cell under conditions that allow theexpression and replication of the infective clone and the recovery ofthe virions obtained from the recombinant coronavirus, which contain theinfective genome of the coronavirus. The infective clone of theinvention can be introduced into the host cell in various ways, forexample by transfection of the host cell with an RNA transcribed invitro from an infective clone of the invention, or by infecting the hostcell with the infective cDNA clone of the invention. Said host cellsthat contain the infective clone of the invention constitute anadditional object of the present invention.

[0103] The invention also provides a set of recombinant viral vectorsderived from an infective clone of the invention, henceforth viralvectors of-the invention. The viral vectors of the invention comprise aninfective cDNA clone of the invention modified to contain a heterologousnucleic acid inserted into said infective clone under conditions thatallow said heterologous nucleic acid to be expressed.

[0104] The term “nucleic acid,” as it is used in this description,includes genes or gene fragments as well as, in general, any molecule ofDNA or RNA.

[0105] In the sense used in this description, the term “heterologous”applied to a nucleic acid refers to a nucleic acid sequence that is notnormally present in the vector used to introduce the heterologousnucleic acid into a host cell.

[0106] The heterologous nucleic acid that can contain the viral vectorof the invention can be a gene or fragment that codes a protein, apeptide, an epitope, or any gene product of interest (such asantibodies, enzymes, etc.). The heterologous nucleic acid can beinserted into the infective clone of the invention by means ofconventional genetic engineering techniques in any appropriate region ofthe cDNA, for example, after ORF 1b or between genes N and 7, followingthe initiator codon (AUG), and in reading frame with that gene; or,alternatively, in the zones corresponding to other ORFs. In theconstruction of the viral vector of the invention, it is essential thatthe insertion of the heterologous nucleic acid not interfere with any ofthe basic viral functions.

[0107] The viral vector of the invention can express one or moreactivities. In this latter case, the viral vector will include as manysequences of heterologous nucleic acid as activities to be expressed,preceded by one or several promoters, or by a promoter and variousribosome recognition sites (IRES, internal ribosome entry sites), or byvarious promoters and one ribosome recognition site.

[0108] Therefore, the invention provides a method for producing aproduct of interest that comprises cultivating a host cell that containsa viral vector of the invention under conditions that allow theheterologous nucleic acid to be expressed and the product of interest tobe recovered. Said host cells that contain the viral vector of theinvention constitute an additional object of the present invention.

[0109] The viral vector of the invention can be designed so that itsspecies specificity and tropism can be easily controlled. Due to thesecharacteristics, a very interesting application of the viral vectors ofthe invention is their use in gene therapy as a vector of the gene ofinterest, or as a vaccinal vector to induce immune responses againstdifferent pathogens.

[0110] The invention furthermore provides vaccines, capable ofprotecting an animal against the infection caused by an infectiousagent, that comprise (i) at least one viral vector of the invention thatexpresses at least one antigen suitable for inducing an immune responseagainst said infectious agent, or an antibody that provides protectionagainst said infectious agent, along with, optionally, (ii) apharmaceutically acceptable excipient.

[0111] In the sense used in this description, “inducing protection”should be understood as the immune response of the receiving organism(animal to be immunized) induced by the viral vector of the invention,through suitable mechanisms such as that induced by substances thatpotentiate cellular response (interleukins, interferons, etc.), cellularnecrosis factors, and similar substances that protect the animal frominfections caused by infectious agents.

[0112] Included under the term “animal” are all animals of any species,preferably mammals, including man.

[0113] The term “infectious agent” in the sense used in this descriptionincludes any viral, bacterial, fungal, parasitic, or other infectiveagent that can infect an animal and cause it a pathology.

[0114] In one particular realization, the vaccine provided by thisinvention comprises at least one viral vector of the invention thatexpresses at least one antigen capable of inducing a systemic immuneresponse and/or an immune response in mucous membranes against differentinfectious agents that propagate in respiratory or intestinal mucousmembranes. The vectors of the invention are quite suitable to induceimmunity in mucous membranes as well as lactogenic immunity, which is ofspecial interest in protecting newborns against intestinal tractinfections.

[0115] In another particular realization, the vaccine provided by thisinvention comprises at least one viral vector of the invention thatexpresses at least one gene that codes for the light and heavy chains ofan antibody of any isotype (for example, IgG₁, IgA, etc.) that protectsagainst an infectious agent.

[0116] Species specificity can be controlled so that the viral vectormay express the S protein of the envelope of a coronavirus that infectsthe desired species (man, dog, cat, pig, etc.), suitable to berecognized by the cellular receptors of the corresponding species.

[0117] The vaccines provided by this invention can be monovalent ormultivalent, depending on whether the viral vectors of the inventionexpress one or more antigens capable of inducing an immune response toone or more infectious agents, or one or more antibodies that provideprotection against one or more infectious agents.

[0118] In a particular realization of this invention, monovalentvaccines capable of protecting man, pigs, dogs and cats againstdifferent infectious human, porcine, canine, and feline agents areprovided, and tropism is controlled by expressing the S glycoprotein ofthe coronavirus with the desired species specificity.

[0119] The monovalent vaccines against porcine infectious agents cancontain a vector that expresses an antigen selected from the groupconsisting essentially of antigens of the following porcine pathogens:Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilusparasuis, porcine parvovirus, Leptospira, Escherichia coli,Erysipelotrix rhusiopathiae, Pasteurella multocida, Bordetellabronchiseptica, Clostridium sp., Serpulina hydiosenteriae, Mycoplasmahyopneumoniae, porcine epidemic diarrhea virus (PEDV), porcinerespiratory coronavirus, rotavirus, or against the pathogens that causeporcine respiratory and reproductive syndrome, Aujeszky's disease(pseudorabies), swine influenza, or transmissible gastroenteritis, andthe etiological agent of atrophic rhinitis and proliferative ileitis.The monovalent vaccines against canine infectious agents can contain anexpression vector that expresses an antigen selected from the groupessentially consisting of antigens of the following canine pathogens:canine herpes viruses, types 1 and 2 canine adenovirus, types 1 and 2canine parvovirus, canine reovirus, canine rotavirus, caninecoronavirus, canine parainfluenza virus, canine influenza virus,distemper virus, rabies virus, retrovirus, and canine calicivirus.

[0120] The monovalent vaccines against feline infectious agents cancontain an expression vector that expresses an antigen selected from thegroup essentially consisting of antigens of the following felinepathogens: cat calicivirus, feline immunodeficiency virus, feline herpesviruses, feline panleukopenia virus, feline reovirus, feline rotavirus,feline coronavirus, cat infectious peritonitis virus, rabies virus,feline Chlamydia psittaci, and feline leukemia virus.

[0121] The vectors can express an antibody that provides protectionagainst an infectious agent, for example, a porcine, canine or felineinfectious agent such as those cited above. In one particularrealization, the vector expresses the recombinant monoclonal antibodyidentified as 6A.C3, which neutralizes TGEV, expressed with isotypesIgG, or IgA, in which the constant part of the immunoglobulin is ofporcine origin, or neutralizing antibodies for human and porcinerotaviruses.

[0122] As the excipient, a diluent such as physiological saline or othersimilar saline solutions can be used. Likewise, these vaccines can alsocontain an adjuvant from those usually used in the formulation of bothaqueous vaccines, such as aluminum hydroxide, QuilA, suspensions ofalumina gels and the like, and oily vaccines based on mineral oils,glycerides, fatty acid derivatives, and their mixtures.

[0123] The vaccines of the present invention can also containcell-response-potentiating (CRP) substances, that is, substances thatpotentiate subpopulations of helper T-cells (Th₁ and Th₂) such asinterleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-6, IL-12, gamma-IFN(gamma-interferon), cellular necrosis factor, and similar substancesthat could theoretically provoke cellular immunity in vaccinatedanimals. These CRP substances could be used in vaccine formulations withaqueous or oily adjuvants. Another type of adjuvants that modulate andimmunostimulate cellular response can also be used, such as MDP (muramyldipeptide), ISCOM (Immunostimulant Complex), or liposomes.

[0124] The invention provides multivalent vaccines capable of preventingand protecting animals from infections caused by different infectiousagents. These multivalent vaccines can be prepared from viral vectors ofthe invention into which the different sequences that code thecorresponding antigens have been inserted in the same recombinantvector, or by constructing independent recombinant vectors that wouldlater be mixed for joint inoculation. Therefore, these multivalentvaccines comprise a viral vector that contains more than one sequence ofheterologous nucleic acids that code for more than one antigen or,alternatively, different viral vectors, each of which expresses at leastone different antigen.

[0125] Analogously, multivalent vaccines that comprise multivalentvectors can be prepared using sequences that code antibodies thatprotect against infectious agents, instead of sequences that code theantigens.

[0126] In one particular realization of this invention, vaccines capableof immunizing humans, pigs, dogs, and cats against different porcine,canine and feline infectious agents, respectively, are provided. Forthis, the viral vectors contained in the vaccine must express differentantigens of the human, porcine, canine or feline pathogens mentionedabove or others.

[0127] The vaccines of this invention can be presented in liquid orlyophilized form and can be prepared by suspending the recombinantsystems in the excipient. If said systems were in lyophilized form, theexcipient itself could be the reconstituting substance.

[0128] Alternatively, the vaccines provided by this invention can beused in combination with other conventional vaccines, either formingpart of them or as a diluent or lyophilized fraction to be diluted withother conventional or recombinant vaccines.

[0129] The vaccines provided by this invention can be administered tothe animal orally, nasally, subcutaneously, intradermally,intraperitoneally, intramuscularly, or by aerosol.

[0130] The invention also provides a method for the immunization ofanimals, in particular pigs, dogs and cats, against one or variousinfectious agents simultaneously, that comprises the oral, nasal,subcutaneous, intradermal, intraperitoneal, intramuscular, or aerosoladministration (or combinations thereof) of a vaccine that contains animmunologically efficacious quantity of a recombinant system provided bythis invention.

[0131] In addition, the invention also provides a method for protectingnewborn animals against infectious agents that infect said animals,consisting in the oral, nasal, subcutaneous, intradermal,intraperitoneal, intramuscular, or aerosol administration (orcombinations thereof) of a vaccine of those provided by this inventionto mothers before or during the gestation period, or to their offspring.

[0132] The invention is illustrated by the following examples, whichdescribe in detail the obtainment of infective clones and theconstruction of the viral vectors of the invention. These examplesshould not be considered as limiting the scope of the invention, but asillustrating it. In said example, the transformation and growth ofbacteria, DNA purification, sequence analysis, and the-assay to evaluatethe stability of the plasmids were carried out according to themethodology described below.

[0133] Transformation of Bacteria

[0134] All of the plasmids were electroporated in the E. coli DH10Bstrain (Gibco BRL), introducing slight modifications to previouslydescribed protocols (Shizuya et al., 1992). For each transformation, 2μL of the ligation and 50 μL of competent bacteria were mixed in 0.2-cmdishes (BioRad) and electroporated at 200 Ω, 2.5 kV, and 25 μF. Then, 1mL of SOC medium (Maniatis et al., 1989) was added at eachtransformation, the cells were incubated a 37° C. for 45 min, andfinally, the recombinant colonies were detected on plates of LB SOCmedia (Maniatis et al., 1989) with 12.5 μg/mL of chloramphenicol.

[0135] Growth Conditions of the Bacteria

[0136] The bacteria containing the original plasmids, in which theincomplete genome of TGEV was cloned (FIG. 3), were grown at 37° C.,showing normal growth kinetics. On the other hand, the BAC thatcontained the complete cDNA was grown at 30° C. for the purpose ofminimizing instability as much as possible. Even so, the size of thecolonies was reduced and incubation periods of up to 24 h were necessaryto achieve normal colony sizes.

[0137] Purification of DNA

[0138] The protocol described by Woo (Woo et al., 1994) was followed,with slight modifications. From a single colony, 4 L of LB wereinoculated with chloramphenicol (12.5 μg/ml). After an incubation periodof 18 h at 30° C., the bacteria were collected by centrifugation at6,000 G, and the plasmid was purified using the Qiagen PlasmidMaxipreparations kit according to the manufacturer's recommendations. Bymeans of this procedure, it was observed that the plasmid DNA obtainedwas contaminated with bacterial DNA. To eliminate the contaminatingbacterial DNA, the plasmidic DNA was purified by means of centrifugationat 55,000 rpm for 16 h on a CsCl gradient. The yield obtained wasbetween 15 and 30 μg/L, depending on the size of the plasmid.

[0139] Sequence Analysis

[0140] The DNA was sequenced in an automatic sequencer (373 DNASequencer, Applied Biosystems) using dideoxynucleotides marked withfluorochromes and a temperature-resistant polymerase (Perkin Elmer). Thereagents were obtained by way of a kit (ABI PRISM Dye Terminator CycleSequencing Ready Reaction Kit) from the Applied Biosystems company. Thethermocycler used to perform the sequencing reactions was a “GeneAmpPCRSystem 9600” (Perkin Elmer).

[0141] The joining of the sequences and their comparison with theconsensus sequence of the TGEV were carried out using the SeqMan II andAlign (DNASTAR) programs, respectively. No differences in relation tothe consent sequence were detected.

[0142] Stability of the Plasmids

[0143] From the original glycerolates, the bacteria that containedrecombinant pBeloBAC11 plasmids were grown in 20 mL of LB withchloramphenicol (12.5 μg/mL) for 16 h at 30° C. and 37° C. This materialwas considered passage 0. The bacteria were diluted 10⁶ times and grownat 30° C. and 37° C. for 16 h. Serial passages were realized duringeight consecutive days (each passage represents approximately 20generations). The plasmid DNA was purified by Miniprep at passages 0 and8 (160 generations) and analyzed with restriction endonucleases. The twoplasmids that contained part of the genome of TGEV were highly stable,whereas the plasmid that contained the complete genome of TGEV showed acertain instability after 40 generations (at this point approximately80% of the DNA presented the correct restriction pattern).

EXAMPLE 1

[0144] Construction of a Recombinant Vector based on a Clone ofInfective cDNA Derived from TGEV

[0145] 1.1 Generation of an Infective cDNA of TGEV

[0146] For the purpose of obtaining a cDNA that coded for the completeTGEV genome, we originally started with a cDNA that coded for thedefective DI-C genome (Mendez et al., 1996). This cDNA, with anapproximate length of one third of the TGEV genome, was cloned in thelow-copy pACNR1180 plasmid (Ruggli et al., 1996) and its sequence wasdetermined. The cDNA that coded the defective genome was efficientlyrescued (replicated and packaged) with the help of a complementing virus(Mendez et al., 1996; Izeta et al., 1999).

[0147] The DI-C genome presents three deletions (Δ1, Δ2, and Δ3) ofapproximately 10, 1 and 8 kilobases (kb), at ORFs 1a, 1b, and betweengenes S and 7, respectively (see FIG. 1).

[0148] The strategy followed to complete the sequence of a cDNA thatwould code for an infective TGEV genome was to incorporate, step bystep, the sequences deleted in the DI-C genome, analyzing thebacteriotoxicity of the new generated constructions. This aspect is veryimportant, since it is widely documented in the scientific literaturethat recombinant plasmids presenting cDNAs of RNA virus generally grewpoorly and were unstable (Boyer and Haenni, 1994; Rice et al., 1989;Mandl et al., 1997).

[0149] The first deletion to be completed was deletion Δ2, of 1 kb, ofORF 1b, yielding a stable recombinant plasmid. The sequence that lackedORF 1a was introduced by cloning cDNA fragments A, B, C, and D (FIG. 1)(Almazan et al., 2000) in such a way that all the information requiredfor the gene of the replicase would be complete. The recombinant plasmidobtained was unstable in the bacteria, generating new plasmids that hadincorporated additions and deletions in fragment B (Almazan et al.,2000). Interestingly, the elimination of a 5,198 bp ClaI-ClaIrestriction fragment that encompassed the region of the genome comprisedbetween nucleotides 4,417 and 9,615 (Penzes et al., 1999) yielded arelatively stable plasmid in the E. coli DH10B strain. Later, thesequence of deletion A3 was added by cloning all the genetic informationfor the structural and nonstructural proteins of the 3′-end of the TGEVgenome (FIG. 1).

[0150] For the purpose of incrementing the stability of the TGEV cDNA,it was decided that it would be subcloned in BAC using the pBeloBAC11plasmid (Kim et al., 1992) (see FIG. 2). The pBeloBAC11 plasmid was agenerous gift from H. Shizuya and M. Simon (California Institute ofTechnology). The plasmid, 7,507 bp in size, includes the replicationorigin of the F factor from parB, parC, E. coli (oriS) and the genesnecessary to keep a single copy of the plasmid per cell (parA, andrepE). The plasmid also presents the gene of resistance tochloramphenicol (cat) as a selection marker. The cDNA was cloned underthe control of the IE promoter of CMV, due to the high level ofexpression obtained using this promoter (Dubensky et al., 1996) and toprevious results obtained in our laboratory, indicating that large (9.7kb and 15 kb) defective genomes derived from TGEV expressed RNAs thatdid not undergo splicing during transport from the nucleus, where theyare synthesized, to the cytoplasm (Izeta et al., 1999; Penzes et al.,1999; Almazan et al., 2000). The generated TGEV cDNA (pBAC-TcDNA-AClaI)contained the information for the genes of the replicase, with theexception of the deleted 5,198 bp ClaI fragment, and all the informationof the structural and nonstructural genes. The 3′-end of the cDNAappears flanked by a 24 nt polyA sequence, the HDV ribozyme, and thetranscription termination sequence of BGH (Izeta et al., 1999). On theother hand, the ClaI fragment necessary to generate a complete genome ofTGEV was cloned in BAC, generating the plasmid pBAC-B+C+D5′, whichcontained the region of the TGEV genome between 4,310 and 9,758 (seeFIG. 3). Both plasmids were grown in the E. coli DH10B strain andsequenced in their entirety. The sequence obtained was identical to theconsent sequence of the PUR46-MAD isolate of TGEV provided at the end ofthis document (SEQ ID NO: 1), with the exception of two replacements inthe positions of nucleotides 6,752 (A=>G, silent) and 18,997 (T=>C,silent), and the changes in the S gene of the PUR46-MAD that has beenreplaced by the D gene of isolate C11 (these changes are indicated inFIG. 4).

[0151] Furthermore, for the purpose of generating a cDNA that would codea virulent TGEV, the S gene of the PUR46-MAD isolate, which replicatesat highs levels in the respiratory tract (>10⁶ PFU/g of tissue) and atlow levels in the intestinal tract (<10³ PFU/mL), was completelyreplaced by the S gene of TGEV clone 11, henceforth C11, whichreplicates with elevated titers both in the respiratory tract (<10PFU/mL) and in the intestinal tract (<10 PFU/mL) (Sanchez et al., 1999).The S gene of C11 presents 14 nucleotides that differ from the S gene ofthe PUR46-MAD isolate, plus a 6 nt insertion at the 5′-end of the S gene(see FIG. 4) (Sanchez et al., 1999). Previous results in our laboratory(Sanchez et al., 1999) showed that mutants generated by directedrecombination, in which the S gene of the PUR46-MAD isolate of the TGEVwas replaced with the S gene of the C11 intestinal isolate, acquiredintestinal tropism and increased virulence, unlike the natural PUR46-MADisolate of the TGEV that replicates very little or not at all in theintestinal tracts of infected pigs.

[0152] A cDNA was constructed from the PUR46-MAD isolate of TGEV withthe S gene of the intestinal isolate C11, by means of cloning of the5,198 bp ClaI-ClaI fragment, obtained from the pBAC-B+C+D5′ plasmid, inthe pBAC-TcDNA^(−ΔClaI) plasmid, to generate the pBAC-TcDNA^(FL) plasmidthat contains the cDNA that codes for the complete TGEV genome (FIG. 3).

[0153] The stability in bacteria of the plasmids used in theconstruction of the clone of infective cDNA (pBAC-TcDNA^(−ΔClaI) andpBAC-ClaI^(F)), as well as the plasmid that contains the complete cDNA(pBAC-TcDNA^(FL)); was analyzed after being grown in E. coli for 160generations. The stability was analyzed by means of digestion withrestriction enzymes of the purified DNAs. No deletions or insertionswere detected, although the presence of minor changes not detected bythe analysis technique used cannot be ruled out in the case of thepBAC-TcDNA^(−ΔclaI) plasmid and the pBAC-B+C+D5′ plasmid. In the case ofthe pBAC-TcDNA plasmid, which contains the complete genome of TGEV, acertain instability was detected after 40 generations (at this pointapproximately 80% of the DNA presented the correct restriction pattern).This slight instability, however, does not represent an obstacle to therescue of the infective virus, since 20 generations (4 L of culture) ofbacterial growth are sufficient to generate a quantity of plasmid DNAthat allows the virus to be rescued.

[0154] 1.2 Rescue of an Infective TGEV from a cDNA that Codes for theComplete Genome

[0155] ST cells were transfected with the pBAC-TcDNA^(FL) plasmid. At 48h posttransfection, the supernatant of the culture was collected andpassed into ST cells six times (see FIG. 5). Starting at passage 2, at14 h postinfection, the cytopathic effect became apparent, extendinglater, at 20 h postinfection, to practically all of these cells thatformed the monolayer (see FIG. 6). On the other hand, the titer ofrescued virus increased rapidly with the passages, reaching values onthe order of 10⁸ PFU/mL as of passage 3 (see FIG. 7). The experiment wasrepeated five times, and in ail cases, infective virus with similartiters were recovered, whereas, in the case of nontransfected ST cellsor ST cells transfected with a similar plasmid, where the ClaI-ClaIfragment was found in the opposite orientation, virus was neverrecovered.

[0156] For the purpose of eliminating the possibility that the virusobtained was the product of contamination, the sequence at positions6,752 and 18,997 was determined by means of sequencing of cDNA fragmentsamplified by RT-PCR using the genomic RNA of the rescued virus as atemplate. The analysis of the sequence determined that the nucleotidesin positions 6,752 and 18,997 were those present in the cDNA.Furthermore, the rescued virus presented, in the cDNA sequence of the Sgene, a restriction site DraIII at position 20,990, as was expected forthe S gene of C11 (FIG. 8). The presence of these three genetic markersconfirmed that the isolated virus came from the cDNA.

[0157] In a more in-depth characterization of the virus generated, acomparative analysis was made by immunofluorescence of infected cellswith the virus recovered (TcDNA) after transfection with the pBAC-TcDNAplasmid or cells infected with the PUR46-MAD isolate of the TGEV. Forthis, specific polyclonal and monoclonal antibodies that recognized boththe C11 isolate and the PUR46-MAD isolate, or only the latter, were used(see FIG. 10). The results obtained confirmed the antigenicity expectedfor the new TcDNA virus. The polyclonal antibody specific for TGEV, theexpected specific monoclonal of the S protein (ID.B12 and 6A.C3), aswell as the specific monoclonal of the M (3B.B3) and N (3B.D8) proteinsrecognized both the TcDNA and the PUR46-MAD. The data obtained indicatedthat the virus generated presented the M and N proteins of the PUR46-MADisolate and the S protein of the C11 isolate, as had been designed inthe original cDNA.

[0158] 1.3 In Vivo Infectivity and Virulence

[0159] For the purpose of analyzing the in vivo, infectivity of theTcDNA virus, a group of five newborn pigs was inoculated with viruscloned from passage 6, and mortality was analyzed. The five inoculatedpigs died 3 to 4 days postinoculation, indicating that the TcDNA viruswas virulent. In contrast, two pigs inoculated only with the diluent ofthe virus and maintained in the same conditions did not sufferalterations.

[0160] 1.4 Optimization of the Levels of Expression by Modification ofthe Transcription-Regulating Sequences

[0161] RNA synthesis in coronavirus takes place by means, of anRNA-dependent process, in which the mRNAs are transcribed from templateswith negative polarity. In the TGEV, a conserved consensus sequence,CUAAAC, appears, which is located just in front of the majority of thegenes. These sequences represent signals for the transcription of thesubgenomic mRNAs. In coronavirus, there are between six and eight typesof mRNAs with variable sizes, depending on the type of coronavirus andof the host. The largest corresponds to the genomic RNA, which in turnserves as mRNA for ORFs 1a and 1b. The rest of the mRNAs correspond tosubgenomic mRNAs. These RNAs are denominated mRNA 1 to 7, in decreasingsize order. On the other hand, some mRNAs that have been discoveredafter the set of originally described mRNAs have been denominated withthe name of the corresponding mRNA, a dash, and a number, e.g., mRNA2-1. The mRNAs present a coterminal structure in relation to thestructure of the genomic RNA. With the exception of the smallest mRNA,the rest are structurally polycistronic, while, in general, only the ORFlocated closest to 5′ is translated.

[0162] The efficiency in the expression of a marker gene (GUS) has beenstudied using different sequences flanking the 5′-terminal of theminimal intergenic (IG) sequence CUAAAC (FIG. 11), different sequencesflanking the 3′-terminal of the IG sequence (FIG. 12), and variousinsertion sites (FIG. 13). The results obtained (FIGS. 11 to 13)indicated that optimal expression was achieved with a TRS consisting of:(i) the −88 nt flanking the consent sequence for the N gene of TGEV;(ii) the IG sequence; and (iii) the 3′-flanking sequence of the IGsequence of the S gene. Furthermore, in agreement with the resultsobtained in relationship to the point of insertion of the heterologousgene, the greatest levels of expression were achieved when theheterologous gene was located at the 3′-end of the genome. A TRS such asthat described allows the GUS to be expressed at levels between 2 and 8μg per 10⁶ cells.

[0163] 1.5 Tissue Specificity of the System of Expression

[0164] Many pathogens enter the host through the mucous membranes. Toprevent this type of infections, it is important to develop systems ofexpression that allow the induction of high levels of secretoryimmunity. This can be achieved fundamentally through the administrationof antigens in the lymph nodes associated with the respiratory andintestinal tract. To achieve this goal, and in general to direct theexpression of a gene at the tissue of interest, the molecular bases ofthe tropism of TGEV have been studied. These studies have showed thatthe tissue specificity of TGEV can be modified by the construction ofrecombinant viruses containing the S gene of coronavirus with thedesired tropism (Ballesteros et al., 1997; Sanchez et al., 1999). Thisinformation makes it possible to construct systems of expression basedon cDNA genomes of coronavirus with respiratory or intestinal tropism.

[0165] 1.6 Expression of the Viral Antigen Coded by the ORF5 of PRRSVusing Infective cDNA

[0166] For the purpose of optimizing the levels of expression ofheterologous genes, constructions were made from a vector ofinterchangeable modules flanked by cloning sequences that facilitate theexchange of TRSs and heterologous genes within the vector. Theconstruction, which included ORF 5 of the PRRSV (Porcine respiratory andreproductive syndrome virus) flanked at the 5′-end by the minimal IGSconsensus sequence (CUAAAC) preceded by the −88 nts flanking the gene ofthe viral nucleocapsid (N), and at the 3′-end by restriction site SalI(GTCGAC) and a sequence analogous to that of Kozak (AC)GACC, yielded anoptimal expression (about 10 μg/10⁶ cells). In principle, these levelsof expression of the heterologous gene are more than sufficient toinduce an immune response. The heterologous gene was inserted into theposition previously occupied by genes 3a and 3b of the virus, which aredispensable.

[0167] 1.7 Induction of an Immune Response in Swine to an AntigenExpressed with the cDNA Derived Virus Vector

[0168] Using the same type of virus vector derived from the cDNA and theTRSs described above, the gene encoding the green fluorescent protein(GFP) was expressed at high levels (20 μg of protein per million ofcells in swine testis, ST, cells). The expression levels were stable formore than 20 passages in cell culture. Furthermore, a set of swine wereimmunized with the live virus vector, that was administered by the oral,intranasal and intragastric routes and a strong humoral immune responsewas detected against both the virus vector and the GFP. Interestingly,no secondary effect was observed in the inoculated animals after theadministration of three doses of the virus vector.

[0169] 1.8 Construction of a Safe Virus Vector that Expresses theForeign Gene without Leading to the Generation of an Infectious Virus.

[0170] To design vector for humans, biosafety is a priority. To achievethis goal, three types of safety guards are being engineered in thevector. Two of these are based on the deletion of two virus components,mapping at different positions of the virus genome, essential for thereplication of the virus. These components are being provided in transby a packaging cell line. This cell (Baby Hamster Kidney, BHK) expressesthe missing TGEV genes encoding essential structural proteins of thevirus (the envelope E and the membrane M proteins). The third safetyguard is the relocation of the packaging signal of the virus genome, insuch a way that the recovery of an infectious virus by recombination isprevented, leading to the generation of a suicide vector thatefficiently expresses the heterologous genes but that is unable topropagate even to the closest neighbor cell.

[0171] With the design of the new vector for use in humans, we are notproducing a new virus that could be propagated within the human species,since this vector can not be transmitted from cell to cell in humanbeings. The vector is based on a replication defective virus. It canonly be grown in the vaccine factory by using packaging cellscomplementing the deletions of the virus. These safety guards representnovel procedures in the engineering of coronaviruses. The recombinantvirus with a new tropism will be replication competent at least infeline cells, since these cells replicate human, porcine, canine andfeline coronaviruses.

[0172] Deposition of Microorganisms:

[0173] The bacterium derived from Escherichia coli, carrying the plasmidwith the infective clone of the invention, identified as Escherichiacoli pBAC-TcDNA^(FL), has been deposited with the Spanish Collection ofType Cultures (CECT), Burjassot (Valencia), on Nov. 24^(th) 1999, underregistration number CECT 5265.

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0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO 1<211> LENGTH: 28588 <212> TYPE: DNA <213> ORGANISM: PorcineTransmissible Gastroenteritis V <400> SEQUENCE: 1 acttttaaag taaagtgagtgtagcgtggc tatatctctt cttttacttt aactagcctt 60 gtgctagatt ttgtcttcggacaccaactc gaactaaacg aaatatttgt ctttctatga 120 aatcatagag gacaagcgttgattatttcc attcagtttg gcaatcactc cttggaacgg 180 ggttgagcga acggtgcagtagggttccgt ccctatttcg taagtcgcct agtagtagcg 240 agtgcggttc cgcccgtacaacgttgggta gaccgggttc cgtcctgtga tctccctcgc 300 cggccgccag gagaatgagttccaaacaat tcaagatcct tgttaatgag gactatcaag 360 tcaacgtgcc tagtcttcctattcgtgacg tgttacagga aattaagtac tgctaccgta 420 atggatttga gggctatgttttcgtaccag aatactgtcg tgacctagtt gattgcgatc 480 gtaaggatca ctacgtcattggtgttcttg gtaacggagt aagtgatctt aaacctgttc 540 ttcttaccga accctccgtcatgttgcaag gctttattgt tagagctaac tgcaatggcg 600 ttcttgagga ctttgaccttaaaattgctc gcactggcag aggtgccata tatgttgatc 660 aatacatgtg tggtgctgatggaaaaccag tcattgaagg cgattttaag gactacttcg 720 gtgatgaaga catcattgaatttgaaggag aggagtacca ttgcgcttgg acaactgtgc 780 gcgatgagaa accgctgaatcagcaaactc tctttaccat tcaggaaatc caatacaatc 840 tggacattcc tcataaattgccaaactgtg ctactagaca tgtagcacca ccagtcaaaa 900 agaactctaa aatagttctgtctgaagatt acaagaagct ttatgatatc ttcggatcac 960 cctttatggg aaatggtgactgtcttagca aatgctttga cactcttcat tttatcgctg 1020 ctactcttag atgcccgtgtggttctgaaa gtagcggcgt tggagattgg actggtttta 1080 agactgcctg ttgtggtctttctggcaaag ttaagggtgt cactttgggt gatattaagc 1140 ctggtgatgc tgttgtcactagtatgagcg caggtaaggg agttaagttc tttgccaatt 1200 gtgttcttca atatgctggtgatgttgaag gtgtctccat ctggaaagtt attaaaactt 1260 ttacagttga tgagactgtatgcacccctg gttttgaagg cgaattgaac gacttcatca 1320 aacctgagag caaatcactagttgcatgca gcgttaaaag agcattcatt actggtgata 1380 ttgatgatgc tgtacatgattgtatcatta caggaaaatt ggatcttagt accaaccttt 1440 ttggtaatgt tggtctattattcaagaaga ctccatggtt tgtacaaaag tgtggtgcac 1500 tttttgtaga cgcttggaaagtagtagagg agctttgtgg ttcactcaca cttacataca 1560 agcaaattta tgaagttgtagcatcacttt gcacttctgc ttttacgatt gtaaactaca 1620 agccaacatt tgtggttccagacaatcgtg ttaaagatct tgtagacaag tgtgtgaaag 1680 ttcttgtaaa agcatttgatgtttttacgc agattatcac aatagctggt attgaggcca 1740 aatgctttgt gcttggtgctaaatacctgt tgttcaataa tgcacttgtc aaacttgtca 1800 gtgttaaaat ccttggcaagaagcaaaagg gtcttgaatg tgcattcttt gctactagct 1860 tggttggtgc aactgttaatgtgacaccta aaagaacaga gactgccact atcagcttga 1920 acaaggttga tgatgttgtagcaccaggag agggttatat cgtcattgtt ggtgatatgg 1980 ctttctacaa gagtggtgaatattatttca tgatgtctag tcctaatttt gttcttacta 2040 acaatgtttt taaagcagttaaagttccat cttatgacat cgtttatgat gttgataatg 2100 ataccaaaag caaaatgattgcaaaacttg gttcatcatt tgaatatgat ggtgatattg 2160 atgctgctat tgtaaaagtcaatgaactac tcattgaatt taggcagcaa agcttgtgct 2220 tcagagcttt taaggacgacaaaagcattt ttgttgaagc ctattttaaa aagtataaaa 2280 tgccagcatg ccttgcaaaacatattggtt tgtggaacat cataaagaaa gattcatgta 2340 agaggggttt tcttaatctcttcaatcact tgaatgaatt ggaagatatc aaagaaacta 2400 atattcaggc tattaaaaacattctttgcc ctgatcctct tcttgatctg gattatggtg 2460 ccatttggta caattgcatgccaggttgct ctgatccttc agttttgggg agtgttcaac 2520 ttttgatcgg taatggtgtgaaagtagttt gtgatggctg caaaggtttt gctaaccaac 2580 tttcaaaagg ttacaacaagctctgtaatg cggctcgcaa tgatattgag atcggtggta 2640 taccattttc cacttttaaaacacctacaa atacttttat tgaaatgaca gatgctatct 2700 attcagttat tgaacaaggtaaggcattat cctttagaga tgctgatgtg ccagttgtag 2760 acaatggtac catttctactgctgattggt ctgaacccat tctgcttgaa cctgctgaat 2820 atgtaaaacc aaagaacaatggtaatgtca ttgttattgc aggttataca ttttataaag 2880 atgaggatga acatttttatccttatggtt ttggtaaaat tgtgcagaga atgtataata 2940 aaatgggtgg tggtgacaaaactgtctcat tttcagaaga agtagatgtt caagaaattg 3000 cacctgttac acgtgttaaacttgaattcg aatttgacaa tgaaattgta actggtgttc 3060 ttgaacgggc tattggtactagatacaaat ttactggtac aacttgggaa gaatttgaag 3120 agtctatttc tgaagaactcgatgcaatct ttgatactct agcaaaccaa ggtgtcgaac 3180 ttgaaggtta cttcatttatgacacttgtg gtggctttga tataaaaaat ccagatggta 3240 ttatgatctc tcagtatgatatcaatatta ctgctgatga aaaatcagaa gttagtgcat 3300 caagtgaaga agaagaagttgaatctgttg aagaagatcc tgagaatgaa attgtagaag 3360 catctgaagg tgctgaagggacttcttctc aagaagaggt tgaaacagta gaagttgcag 3420 atattacttc tacagaagaagatgttgaca ttgttgaagt atctgctaaa gatgaccctt 3480 gggctgcagc tgttgatgtacaagaagctg aacaatttaa tccttctcta ccacctttca 3540 agacaacgaa tctcaacggaaaaattatcc ttaagcaagg ggataataat tgttggataa 3600 atgcttgttg ctatcagcttcaggcctttg attttttcaa caatgaagct tgggagaaat 3660 ttaagaaagg tgatgtcatggactttgtaa acctttgtta tgcagcaaca acactagcaa 3720 gaggtcattc tggtgatgcagagtatcttc ttgaacttat gctcaatgat tatagcacag 3780 ccaagatagt acttgcagctaagtgtggtt gtggtgaaaa agaaattgtt ttggaaagag 3840 ctgtttttaa actcaccccacttaaggaga gttttaatta tggtgtttgt ggtgactgca 3900 tgcaagttaa cacctgtagatttttaagtg ttgaaggctc tggtgttttt gttcatgaca 3960 tattaagcaa gcaaacgccagaagctatgt ttgttgtcaa acctgttatg catgcagttt 4020 acactggcac aactcaaaatggccattaca tggttgatga tattgaacac ggttattgtg 4080 tagatggtat gggtattaaaccacttaaga aacggtgtta tacatccaca ttgttcatta 4140 atgccaatgt aatgactagagctgaaaaac caaaacaaga gtttaaagtt gaaaaagtag 4200 aacagcaacc gatagtggaggaaaacaaat cctctattga aaaagaggaa attcaaagtc 4260 ctaaaaacga tgaccttatacttccatttt acaaagctgg taaactttcc ttttatcagg 4320 gtgctttgga tgttttgatcaatttcttgg aacctgatgt tattgttaat gctgctaatg 4380 gtgatcttaa acacatgggtggtgtcgcaa gagccatcga tgttttcact ggtggcaaat 4440 taacagaacg ttctaaggattatcttaaaa agaacaaatc tattgctcct ggtaatgctg 4500 ttttctttga aaatgtcattgagcatctta gtgttttgaa tgcagttgga ccacgtaatg 4560 gtgacagccg agttgaagccaaactttgta atgtttacaa agcaattgca aagtgtgaag 4620 gaaaaatatt aacaccacttattagtgttg gtatctttaa tgttagactt gaaacatcat 4680 tgcagtgctt acttaagactgtgaatgaca ggggattgaa tgtcttcgta tacactgacc 4740 aggagaggca aactattgagaatttcttct cttgttctat ccctgtcaat gttactgagg 4800 ataatgttaa ccatgaacgtgtgtctgttt cttttgacaa aacatacggt gaacagctta 4860 agggcaccgt tgtcatcaaagacaaagatg ttacaaacca gttgcctagc gcttttgatg 4920 ttggtcaaaa agttattaaggctattgata tagattggca agctcattat ggtttccgtg 4980 atgctgctgc ttttagcgctagtagtcatg atgcttataa atttgaagtt gttacacata 5040 gcaatttcat tgtgcataagcagactgaca acaactgttg gattaatgca atttgtcttg 5100 cattacagag actcaagccacagtggaaat ttcctggtgt tagaggtctc tggaatgaat 5160 ttcttgagcg taaaacacaaggttttgtac atatgttgta tcacatttct ggagtaaaga 5220 aaggtgagcc aggtgatgctgaattaatgc tgcataaact tggtgacttg atggacaatg 5280 attgtgaaat cattgtcacacacactacag catgtgacaa gtgcgcaaaa gtagaaaagt 5340 ttgttggacc agtggtagcagcacctcttg caattcatgg cactgacgaa acatgtgtgc 5400 atggcgttag tgtcaatgtcaaagtcaccc aaattaaggg cactgttgct attacttctt 5460 tgattggtcc tattattggagaagtactag aagcaactgg ttatatttgt tatagcggtt 5520 ctaacaggaa tggtcattacacctattacg ataaccgtaa tggattagtg gttgatgcag 5580 aaaaggctta ccattttaatagagacttat tacaggtcac aacagctatt gcaagtaatt 5640 tcgttgtcaa gaaaccacaagcagaggaaa gacctaagaa ttgtgctttt aacaaagttg 5700 cagcatctcc taagattgtacaagaacaaa aattgttggc tattgaaagt ggtgctaact 5760 atgctcttac tgaatttggaagatatgctg acatgttctt tatggctgga gataaaattc 5820 ttaggttgct gcttgaagtctttaaatatt tgctggtttt atttatgtgt cttagaagta 5880 ctaagatgcc taaagttaaagtcaaaccac ctcttgcatt taaagatttt ggtgctaagg 5940 tcagaacgct caattacatgagacaattga acaaaccctc tgtctggcgt tacgcaaaac 6000 tagttttatt gttgatagcaatatataatt tcttttattt gtttgtcagt ataccagtag 6060 tgcataaatt aacatgtaacggtgctgtac aggcatataa aaattctagt tttataaagt 6120 ctgcagtctg tggcaactctattttatgca aagcctgttt ggcttcttat gatgagttgg 6180 ctgattttca acatctccaagttacttggg atttcaaatc tgacccacta tggaacagac 6240 tggtacaatt gtcttactttgcattcttgg ctgtttttgg taataactat gttaggtgtt 6300 ttcttatgta ttttgtatctcagtacctca acctttggct ttcttatttt ggttatgtag 6360 agtacagttg gtttttgcatgttgtcaact ttgaatccat ctcagctgag tttgtgatcg 6420 tagttatagt ggttaaggcagttctcgccc ttaaacatat tgttttcgca tgctcaaacc 6480 cgtcttgcaa aacgtgctctaggactgcaa ggcagacacg tattcctatt caagttgttg 6540 ttaatggttc aatgaagactgtttatgttc atgctaatgg tactggtaaa ttctgcaaga 6600 aacacaattt ttattgtaagaactgtgatt cttatggctt tgaaaacaca ttcatctgtg 6660 acgaaattgt acgtgatctcagtaatagtg ttaaacaaac tgtttacgcc actgatagat 6720 ctcatcaaga agtcacaaaagttgaatgtt cagatggctt ttacagattt tatgttggtg 6780 atgaattcac ttcatatgactatgatgtaa aacacaagaa atacagtagt caagaggttc 6840 tcaagagcat gctcttgcttgatgacttca ttgtgtacag tccatctggt tctgctcttg 6900 caaatgttag aaatgcctgtgtttactttt cacaacttat tggtaagcct attaagattg 6960 ttaacagtga tttgcttgaagacctctctg tagattttaa aggggcactt tttaatgcta 7020 aaaagaatgt aattaagaattctttcaatg ttgatgtctc agaatgcaaa aatcttgacg 7080 aatgttacag ggcttgcaatcttaatgttt cattttctac atttgaaatg gctgtcaaca 7140 atgctcatag gtttggtattctgattactg atcgttcttt taacaatttc tggccatcaa 7200 aagttaagcc tggttcatctggtgtgtcgg ccatggacat tggtaagtgt atgacttctg 7260 atgctaagat tgttaatgctaaagttttaa ctcaacgtgg taaaagtgtt gtttggctta 7320 gccaggattt tgctgcacttagctcaactg ctcagaaagt tttggttaaa acttttgtag 7380 aagaaggtgt caacttttcactcacattta atgctgtagg ttcagatgat gatcttcctt 7440 atgaaagatt cactgaatctgtgtctccaa aaagtggttc aggctttttc gatgtaatta 7500 cacagcttaa acaaattgtgattttggttt ttgtttttat ctttatttgt ggtttgtgct 7560 ctgtttacag tgttgctacacagtcctaca ttgaatctgc tgaaggctat gactacatgg 7620 ttattaagaa tggaattgttcaaccttttg acgataccat ttcatgtgtt cataacactt 7680 ataaaggatt cggtgactggtttaaagcta agtatggttt tatccctact tttggtaaat 7740 catgtccaat tgttgtaggaactgtttttg atcttgaaaa tatgagacca attcctgacg 7800 tgcctgcata tgtttctattgtgggtagat ctcttgtttt cgctattaat gctgcttttg 7860 gtgttactaa tatgtgctatgatcatactg gcaatgcagt tagtaaggac tcttactttg 7920 atacttgtgt gtttaatactgcgtgcacca ctcttacagg tcttggtggt acaattgtat 7980 attgtgcaaa gcaaggtttagttgaaggtg ctaagctcta tagtgatctt atgccagact 8040 attattatga gcatgctagtggtaacatgg ttaaattgcc agcaattatt agaggacttg 8100 gtctacgttt tgtgaaaacacaggctacaa cttattgtag agtgggagag tgcattgata 8160 gtaaagctgg tttttgctttggtggcgata actggtttgt ctacgacaat gagtttggca 8220 atggatacat ctgtggtaattctgtgctag gattctttaa gaatgtcttc aaactcttta 8280 actctaacat gtctgtggtagctacatctg gtgcgatgct tgttaacatt attattgcat 8340 gcttagctat tgcaatgtgttatggtgttc ttaagtttaa gaagattttt ggtgattgta 8400 ctttcctcat tgttatgatcattgtcaccc ttgttgtgaa caatgtgtct tattttgtca 8460 ctcaaaacac gttctttatgatcatctacg ccattgttta ctattttata acaagaaaac 8520 ttgcataccc aggcattcttgatgctgggt ttattattgc ttatattaat atggctccat 8580 ggtacgtgat taccgcatatatcctagttt tcctctatga ctcactccct tcactgttta 8640 aacttaaagt ttcaacaaatctttttgaag gtgataaatt tgtgggtaac tttgaatctg 8700 ctgctatggg tacttttgttattgacatgc gttcatatga aactattgtt aattctactt 8760 ctattgctag aattaaatcatatgctaaca gcttcaataa atataagtac tacacaggtt 8820 caatgggaga agctgactacagaatggctt gctatgctca tcttggtaaa gctcttatgg 8880 actattctgt taatagaacagacatgcttt acacacctcc tactgttagt gttaattcta 8940 cacttcagtc aggtttgcggaaaatggcac agcctagtgg tcttgtagag ccttgcattg 9000 taagagtttc ctatggtaacaatgtgctta atggtttatg gttaggagat gaagtcattt 9060 gccctagaca tgttattgctagtgatacca cacgtgttat caactatgaa aatgaaatgt 9120 ctagtgtgag acttcacaacttttcagttt ctaagaataa tgtgtttttg ggtgttgtgt 9180 ctgccagata taagggtgtgaatcttgtac ttaaagtcaa ccaggttaat cctaacacac 9240 cagaacataa atttaagtctattaaagctg gtgaaagttt taacattctt gcttgttatg 9300 aaggatgtcc tggcagtgtttatggtgtca acatgagaag tcaaggtacc attaaaggat 9360 cttttatagc tggtacttgtggatcagtag gttatgtgtt agaaaatgga attctctatt 9420 ttgtatacat gcatcacttagaacttggaa atggctcgca tgttggttcc aattttgaag 9480 gagaaatgta cggtggttatgaagatcaac ctagcatgca attggaaggt actaatgtca 9540 tgtcatcaga taatgtggttgcattcctat atgctgcact tatcaatggt gaaaggtggt 9600 ttgttacaaa cacatcgatgtcattagaat catacaatac atgggccaaa actaacagtt 9660 tcacagaact ttcttcaactgatgctttta gcatgttggc tgcaaaaact ggtcaaagtg 9720 ttgagaaatt actagatagcatcgtaagac tcaacaaggg ttttggaggt cgtactatac 9780 tttcttatgg ctcattgtgtgacgagttca ctccaactga agtcataagg caaatgtatg 9840 gtgtaaatct tcaggctggtaaagtaaaat ctttcttcta ccctattatg actgcaatga 9900 caattctctt tgccttttggcttgaattct ttatgtacac acccttcact tggattaatc 9960 caacttttgt tagcattgtattggctgtta caactttgat ctcgacggtt tttgtctctg 10020 gcatcaaaca taagatgttgttctttatgt cttttgtcct tcctagtgtt atccttgtga 10080 cagcacacaa tttgttctgggacttttctt actatgaaag tcttcagtca attgttgaga 10140 atactaacac tatgtttttgcctgttgaca tgcaaggtgt catgctcaca gtgttttgct 10200 ttattgtctt tgttacatatagtgttagat tcttcacttg caaacaatca tggttctcac 10260 ttgctgtgac aactattcttgtgatcttta acatggttaa aatctttgga acatctgatg 10320 aaccatggac tgaaaaccaaattgctttct gctttgtgaa catgcttact atgattgtca 10380 gtcttactac aaaggattggatggttgtca ttgcatcata cagaattgca tattatattg 10440 ttgtatgtgt aatgccatctgcttttgtat ctgactttgg gtttatgaag tgtattagca 10500 ttgtttacat ggcgtgcggttatttgtttt gttgctatta tggcattctt tattgggtta 10560 acagatttac atgcatgacttgtggtgttt atcaattcac tgtgtctgca gctgaactta 10620 aatacatgac cgctaacaacctttctgcac ctaagaacgc atatgacgct atgattctta 10680 gtgctaaatt gattggtgttggaggtaaga gaaacatcaa aatttcaact gtacagtcaa 10740 aacttacaga gatgaaatgtaccaatgttg tcttgcttgg tcttttatct aaaatgcatg 10800 tcgagtctaa ctcaaaagagtggaactatt gtgttggact acacaatgag ataaaccttt 10860 gtgacgatcc tgaaatcgttcttgagaaac tgttagctct tattgcattc ttcttgtcca 10920 aacataacac ttgtgaccttagcgaactta ttgaatcata ctttgagaac accaccatac 10980 tccagagtgt ggcttcagcttatgctgcat tgcctagctg gattgcactt gaaaaagctc 11040 gcgctgatct tgaagaggctaagaaaaatg atgttagccc tcaaattttg aagcagctta 11100 ctaaagcatt taacattgccaagagtgatt ttgagcgcga agcatcagtg caaaagaaac 11160 tcgacaaaat ggctgagcaggctgcagcta gtatgtataa agaagcacga gctgtggaca 11220 gaaagtcaaa gattgtttctgctatgcata gcctactttt tggtatgctt aagaaacttg 11280 atatgtccag tgtcaacactattattgacc aggctcgtaa tggtgttcta cctttaagta 11340 tcattccagc tgcatcagctacaagacttg ttgttattac acctagcctt gaagtgtttt 11400 ccaagattag gcaagaaaacaatgttcatt atgctggtgc tatttggact attgttgaag 11460 ttaaagatgc taatggttcacatgtacatc ttaaggaagt caccgctgct aatgaattaa 11520 accttacttg gccattgagcattacttgtg agagaaccac aaagcttcag aacaatgaaa 11580 ttatgccagg taaacttaaagaaagagctg tcagagcgtc agcaactctt gatggtgaag 11640 ctttcggcag tggaaaggctcttatggcat ctgaaagtgg aaaaagcttt atgtatgcat 11700 ttatagcctc agacaacaatcttaagtatg ttaagtggga gagcaataat gatattatac 11760 ctattgaact tgaagctccattgcgtttct atgttgacgg cgctaatggt cctgaagtca 11820 agtatttgta ttttgtcaagaatttaaaca ctcttagacg tggtgccgtt cttggttata 11880 tcggtgcaac agttcgtctgcaagctggta aacccactga acatccatct aacagtagtt 11940 tattgacatt gtgtgctttttcacctgatc ctgctaaagc atatgttgat gctgttaaga 12000 gaggcatgca accagttaataactgtgtaa aaatgctctc aaatggtgct ggtaatggta 12060 tggctgttac aaacggtgtcgaagctaaca cacaacagga ctcttatggt ggtgcttcag 12120 tttgtattta ttgcagatgccatgttgaac atcctgctat tgatggatta tgccgctaca 12180 aaggtaagtt cgtgcaaataccaactggca cacaagatcc aattcggttc tgtattgaaa 12240 atgaagtttg tgttgtctgtggttgttggc ttaacaatgg ttgcatgtgc gatcgtactt 12300 ctatgcagag ttttactgttgatcaaagtt atttaaacga gtgcggggtt ctagtgcagc 12360 tcgactagaa ccctgcaatggtactgatcc agaccatgtt agtagagctt ttgacatcta 12420 caacaaagat gttgcgtgtattggtaaatt ccttaagacg aattgttcaa gatttaggaa 12480 tttggacaaa catgatgcctactacattgt caaacgttgt acaaagaccg ttatggacca 12540 tgagcaagtc tgttataacgatcttaaaga ttctggtgct gttgctgagc atgacttctt 12600 cacatataaa gagggtagatgtgagttcgg taatgttgca cgtaggaatc ttacaaagta 12660 cacaatgatg gatctttgttacgctatcag aaattttgat gaaaagaact gtgaagttct 12720 caaagaaata ctcgtgacagtaggtgcttg cactgaagaa ttctttgaaa ataaagattg 12780 gtttgatcca gttgaaaatgaagccataca tgaagtttat gcaaaacttg gacccattgt 12840 agccaatgct atgcttaaatgtgttgcttt ttgcgatgcg atagtggaaa aaggctatat 12900 aggtgttata acacttgacaaccaagatct taatggcaat ttctacgatt tcggcgattt 12960 cgtgaagact gctccgggttttggttgcgc ttgtgttaca tcatattatt cttatatgat 13020 gcctttaatg gggatgacttcatgcttaga gtctgaaaac tttgtgaaaa gtgacatcta 13080 tggttctgat tataagcagtatgatttact agcttatgat tttaccgaac ataaggagta 13140 ccttttccaa aaatactttaagtactggga tcgcacatat cacccaaatt gttctgattg 13200 tactagtgac gagtgtattattcattgtgc taattttaac acattgtttt ctatgacaat 13260 accaatgaca gcttttggaccacttgtccg taaagttcat attgatggtg taccagtagt 13320 tgttactgca ggttaccatttcaaacaact tggtatagta tggaatcttg atgtaaaatt 13380 agacacaatg aagttgagcatgactgatct tcttagattt gtcacagatc caacacttct 13440 tgtagcatca agccctgcacttttagacca gcgtactgtc tgtttctcca ttgcagcttt 13500 gagtactggt attacatatcagacagtaaa accaggtcac tttaacaaag atttctacga 13560 tttcataaca gagcgtggattctttgaaga gggatctgag ttaacattaa aacatttttt 13620 ctttgcacag ggtggtgaagctgctatgac agacttcaat tattatcgct acaatagagt 13680 cacagtactt gatatttgccaagctcaatt tgtttacaaa atagttggca agtattttga 13740 atgttatgac ggtgggtgcattaatgctcg tgaagttgtt gttacaaact atgacaagag 13800 tgctggctat cctttgaacaaatttggtaa agctagactt tactacgaaa ctctttcata 13860 tgaagagcag gatgcactttttgctttaac aaagagaaat gttttaccca caatgactca 13920 aatgaatttg aaatacgctatttctggtaa ggcaagagct cgtacagtag gaggagtttc 13980 acttctttct accatgactacgagacaata tcatcagaag catttgaagt caattgctgc 14040 aacacgcaat gctactgtggtcattggttc aaccaagttt tatggtggtt gggacaatat 14100 gcttaaaaat ttaatgcgtgatgttgataa tggttgtttg atgggatggg actatcctaa 14160 gtgtgaccgt gctttacctaatatgattag aatggcttct gccatgatat taggttctaa 14220 gcatgttggt tgttgtacacataatgatag gttctaccgc ctctccaatg agttagctca 14280 agtactcaca gaagttgtgcattgcacagg tggtttttat tttaaacctg gtggtacaac 14340 tagcggtgat ggtactacagcatatgctaa ctctgctttt aacatctttc aagctgtttc 14400 tgctaatgtt aataagcttttgggggttga ttcaaacgct tgtaacaacg ttacagtaaa 14460 atccatacaa cgtaaaatttacgataattg ttatcgtagt agcagcattg atgaagaatt 14520 tgttgttgag tactttagttatttgagaaa acacttttct atgatgattt tatctgatga 14580 tggagttgtg tgctacaacaaagattatgc ggatttaggt tatgtagctg acattaatgc 14640 ttttaaagca acactttattaccagaataa cgtctttatg tccacttcta agtgttgggt 14700 agaaccagat cttagtgttggaccacatga attttgttca cagcatacat tgcagattgt 14760 tgggcctgat ggagactactatcttcccta tccagacccg tccagaattt tatcagctgg 14820 tgtgtttgtt gatgacatagttaaaacaga caatgttatt atgttagaac gttacgtgtc 14880 attggctatt gacgcatacccgctcacaaa acaccctaag cctgcttatc aaaaagtgtt 14940 ttacactcta ctagattgggttaaacatct acagaaaaat ttgaatgcag gtgttcttga 15000 ttcgttttca gtgacaatgttagaggaagg tcaagataag ttctggagtg aagagtttta 15060 cgctagcctc tatgaaaagtccactgtctt gcaagctgca ggcatgtgtg tagtatgtgg 15120 ttcgcaaact gtacttcgttgtggagactg tcttaggaga ccacttttat gcacgaaatg 15180 tgcttacgac catgttatgggaacaaagca taaattcatt atgtctatca caccatatgt 15240 gtgtagtttt aatggttgtaatgtcaatga tgttacaaag ttgtttttag gtggtcttag 15300 ttattattgt atgaaccacaaaccacagtt gtcattccca ctctgtgcta atggcaacgt 15360 ttttggtcta tataaaagtagtgcagtcgg ctcagaggct gttgaagatt tcaacaaact 15420 tgcagtttct gactggactaatgtagaaga ctacaaactt gctaacaatg tcaaggaatc 15480 tctgaaaatt ttcgctgctgaaactgtgaa agctaaggag gagtctgtta aatctgaata 15540 tgcttatgct gtattaaaggaggttatcgg ccctaaggaa attgtactcc aatgggaagc 15600 ttctaagact aagcctccacttaacagaaa ttcagttttc acgtgttttc agataagtaa 15660 ggatactaaa attcaattaggtgaatttgt gtttgagcaa tctgagtacg gtagtgattc 15720 tgtttattac aagagcacgagtacttacaa attgacacca ggtatgattt ttgtgttgac 15780 ttctcataat gtgagtcctcttaaagctcc aattttagtc aaccaagaaa agtacaatac 15840 catatctaag ctctatcctgtctttaatat agcggaggcc tataatacac tggttcctta 15900 ctaccaaatg ataggtaagcaaaaatttac aactatccaa ggtcctcctg gtagcggtaa 15960 atctcattgt gttataggtttgggtttgta ttaccctcag gcgagaatag tctacactgc 16020 atgttctcat gcggctgtagacgctttatg tgaaaaagca gccaaaaact tcaatgttga 16080 tagatgttca aggataatacctcaaagaat cagagttgat tgttacacag gctttaagcc 16140 taataacacc aatgcgcagtacttgttttg tactgttaat gctctaccag aagcaagttg 16200 tgacattgtt gtagttgatgaggtctctat gtgtactaat tatgatctta gtgtcataaa 16260 tagccgactg agttacaaacatattgttta tgttggagac ccacagcagc taccagctcc 16320 tagaactttg attaataagggtgtacttca accgcaggat tacaatgttg taaccaaaag 16380 aatgtgcaca ctaggacctgatgtcttttt gcataaatgt tacaggtgcc cagctgaaat 16440 tgttaagaca gtctctgcacttgtttatga aaataaattt gtacctgtca acccagaatc 16500 aaagcagtgc ttcaaaatgtttgtaaaagg tcaggttcag attgagtcta actcttctat 16560 aaacaacaag caactagaggttgtcaaggc ctttttagca cataatccaa aatggcgtaa 16620 agctgttttc atctcaccctataatagtca aaattatgtt gctcggcgtc ttcttggttt 16680 gcaaacgcaa actgtggattccgctcaggg tagtgagtat gattacgtca tctacacaca 16740 gacctccgat acacagcatgctactaatgt taacagattt aatgttgcca ttacgagagc 16800 aaaggttggt atactttgtatcatgtgtga tagaactatg tatgagaatc ttgatttcta 16860 tgaactcaaa gattcaaagattggtttaca agcaaaacct gaaacttgtg gtttatttaa 16920 agattgttcg aagagcgaacaatacatacc acctgcttat gcaacgacat atatgagctt 16980 atctgataat tttaagacaagtgatggttt agctgttaac atcggtacaa aagatgttaa 17040 atatgctaat gtcatctcatatatgggatt caggtttgaa gccaacatac caggctatca 17100 cacactattc tgcacgcgagattttgctat gcgtaatgtt agagcatggc ttgggtttga 17160 cgttgaaggt gcacatgtctgtggtgataa tgttggaact aatgtaccat tacagctggg 17220 tttctcaaac ggtgtggattttgtagtgca aactgaagga tgtgttatta ctgaaaaagg 17280 taatagcatt gaggttgtaaaagcacgagc accaccaggt gagcaatttg cacacttgat 17340 tccgcttatg agaaagggtcaaccttggca cattgttaga cgccgtatag tgcagatggt 17400 ctgtgactat tttgatggcttatcagacat tctgatcttt gtgctttggg ctggtggtct 17460 tgaacttaca actatgagatactttgttaa aattggaaga ccacaaaaat gtgaatgcgg 17520 caaaagtgca acttgttatagtagctctca atctgtttat gcttgcttca agcatgcatt 17580 aggatgtgat tatttatataacccttactg cattgacata cagcaatggg gttacacagg 17640 atctttgagc atgaatcatcatgaagtttg caacattcat agaaatgagc atgtagctag 17700 tggtgatgct atcatgactagatgtctcgc tatacatgac tgttttgtca aacgtgttga 17760 ttggtcaatt gtgtacccttttattgacaa tgaagaaaag atcaataaag ctggtcgcat 17820 agtgcagtca catgtcatgaaagctgctct gaagattttt aatcctgctg caattcacga 17880 tgtgggtaat ccaaaaggcatccgttgtgc tacaacacca ataccatggt tttgttatga 17940 tcgtgatcct attaataacaatgttagatg tctggattat gactatatgg tacatggtca 18000 aatgaatggt cttatgttattttggaactg taatgtagac atgtacccag agttttcaat 18060 tgtttgtaga tttgatactcgcactcgctc taaattgtct ttagaaggtt gtaatggtgg 18120 tgcattgtat gttaataaccatgctttcca cacaccagct tatgatagaa gagcttttgc 18180 taagcttaaa cctatgccattcttttacta tgatgatagt aattgtgaac ttgttgatgg 18240 gcaacctaat tatgtaccacttaagtcaaa tgtttgcata acaaaatgca acattggtgg 18300 tgctgtctgc aagaagcatgctgctcttta cagagcgtat gttgaggatt acaacatttt 18360 tatgcaggct ggttttacaatatggtgtcc tcaaaacttt gacacctata tgctttggca 18420 tggttttgtt aatagcaaagcacttcagag tctagaaaat gtggctttta atatcgttaa 18480 gaaaggtgcc ttcaccggtttaaaaggtga cttaccaact gctgttattg ctgacaaaat 18540 aatggtaaga gatggacctactgacaaatg tatttttaca aataagacta gtttacctac 18600 aaatgtagct tttgagttatatgcaaaacg caaacttgga ctcacacctc cattaacaat 18660 acttaggaat ttaggtgttgtcgcaacata taagtttgtg ttgtgggatt atgaagctga 18720 acgtcctttc tcaaatttcactaagcaagt gtgttcctac actgatcttg atagtgaagt 18780 tgtaacatgt tttgataatagtattgctgg ttcttttgag cgttttacta ctacaagaga 18840 tgcagtgctt atttctaataacgctgtgaa agggcttagt gccattaaat tacaatatgg 18900 ccttttgaat gatctacctgtaagtactgt tggaaataaa cctgtcacat ggtatatcta 18960 tgtgcgcaag aatggtgagtacgtcgaaca aatcgatagt tactatacac agggacgtac 19020 ttttgaaacc ttcaaacctcgtagtacaat ggaagaagat tttcttagta tggatactac 19080 actcttcatc caaaagtatggtcttgagga ttatggtttt gaacacgttg tatttggaga 19140 tgtctctaaa actaccattggtggtatgca tcttcttata tcgcaagtgc gccttgcaaa 19200 aatgggtttg ttttccgttcaagaatttat gaataattct gacagtacac tgaaaagttg 19260 ttgtattaca tatgctgatgatccatcttc taagaatgtg tgcacttata tggacatact 19320 cttggacgat tttgtgactatcattaagag cttagatctt aatgttgtgt ccaaagttgt 19380 ggatgtcatt gtagattgtaaggcatggag atggatgttg tggtgtgaga attcacatat 19440 taaaaccttc tatccacaactccaatctgc tgaatggaat cccggctata gcatgcctac 19500 actgtacaaa atccagcgtatgtgtctcga acggtgtaat ctctacaatt atggtgcaca 19560 agtgaaatta cctgtaggcattactactaa gttcgttaag tatactcagt tgtgtcaata 19620 ccttaacact actacattgtgtgtaccaca caaaatgcgt gtattgcatt taggagctgc 19680 tggtgcatct ggtgttgctcctggtagtac tgtattaaga agatggttac cagatgatgc 19740 catattggtt gataatgatttgagagatta cgtttccgac gcagacttca gtgttacagg 19800 tgattgtact agtctttacatcgaagacaa gtttgatttg ctcgtctctg atttatatga 19860 tggctccaca aaatcaattgacggtgaaaa cacgtcgaaa gatggtttct ttacttatat 19920 taatggtttc attaaagagaaactgtcact tggtggatct gttgccatta aaatcacgga 19980 atttagttgg aataaagatttatatgaatt gattcaaaga tttgagtatt ggactgtgtt 20040 ttgtacaagt gttaacacgtcatcatcaga aggctttctg attggtatta actacttagg 20100 accatactgt gacaaagcaatagtagatgg aaatataatg catgccaatt atatattttg 20160 gagaaactct acaattatggctctatcaca taactcagtc ctagacactc ctaaattcaa 20220 gtgtcgttgt aacaacgcacttattgttaa tttaaaagaa aaagaattga atgaaatggt 20280 cattggatta ctaaggaagggtaagttgct cattagaaat aatggtaagt tactaaactt 20340 tggtaaccac ttcgttaacacaccatgaaa aaactatttg tggttttggt cgtaatgcca 20400 ttgatttatg gagacaattttccttgttct aaattgacta atagaactat aggcaaccag 20460 tggaatctca ttgaaaccttccttctaaac tatagtagta ggttaccacc taattcagat 20520 gtggtgttag gtgattattttcctactgta caaccttggt ttaattgcat tcgcaatgat 20580 agtaatgacc tttatgttacactggaaaat cttaaagcat tgtattggga ttatgctaca 20640 gaaaatatca cttggaatcacagacaacgg ttaaacgtag tcgttaatgg atacccatac 20700 tccatcacag ttacaacaacccgcaatttt aattctgctg aaggtgctat tatatgcatt 20760 tgtaagggct caccacctactaccaccaca gaatctagtt tgacttgcaa ttggggtagt 20820 gagtgcaggt taaaccataagttccctata tgtccttcta attcagaggc aaattgtggt 20880 aatatgctgt atggcctacaatggtttgca gatgaggttg ttgcttattt acatggtgct 20940 agttaccgta ttagttttgaaaatcaatgg tctggcactg tcacatttgg tgatatgcgt 21000 gcgacaacat tagaagtcgctggcacgctt gtagaccttt ggtggtttaa tcctgtttat 21060 gatgtcagtt attatagggttaataataaa aatggtacta ccgtagtttc caattgcact 21120 gatcaatgtg ctagttatgtggctaatgtt tttactacac agccaggagg ttttatacca 21180 tcagatttta gttttaataattggttcctt ctaactaata gctccacgtt ggttagtggt 21240 aaattagtta ccaaacagccgttattagtt aattgcttat ggccagtccc tagctttgaa 21300 gaagcagctt ctacattttgttttgagggt gctggctttg atcaatgtaa tggtgctgtt 21360 ttaaataata ctgtagacgtcattaggttc aaccttaatt ttactacaaa tgtacaatca 21420 ggtaagggtg ccacagtgttttcattgaac acaacgggtg gtgtcactct tgaaatttca 21480 tgttatacag tgagtgactcgagctttttc agttacggtg aaattccgtt cggcgtaact 21540 gatggaccac ggtactgttacgtacactat aatggcacag ctcttaagta tttaggaaca 21600 ttaccaccta gtgtcaaggagattgctatt agtaagtggg gccattttta tattaatggt 21660 tacaatttct ttagcacatttcctattgat tgtatatctt ttaatttgac cactggtgat 21720 agtgacgttt tctggacaatagcttacaca tcgtacactg aagcattagt acaagttgaa 21780 aacacagcta ttacaaaggtgacgtattgt aatagtcacg ttaataacat taaatgctct 21840 caaattactg ctaatttgaataatggattt tatcctgttt cttcaagtga agttggtctt 21900 gtcaataaga gtgttgtgttactacctagc ttttacacac ataccattgt taacataact 21960 attggtcttg gtatgaagcgtagtggttat ggtcaaccca tagcctcaac attaagtaac 22020 atcacactac caatgcaggatcacaacacc gatgtgtact gtattcgttc tgaccaattt 22080 tcagtttatg ttcattctacttgcaaaagt gctttatggg acaatatttt taagcgaaac 22140 tgcacggacg ttttagatgccacagctgtt ataaaaactg gtacttgtcc tttctcattt 22200 gataaattga acaattacttaacttttaac aagttctgtt tgtcgttgag tcctgttggt 22260 gctaattgta agtttgatgtagctgcccgt acaagaacca atgagcaggt tgttagaagt 22320 ttgtatgtaa tatatgaagaaggagacaac atagtgggtg taccgtctga taatagtggt 22380 gtgcacgatt tgtcagtgctacacctagat tcctgcacag attacaatat atatggtaga 22440 actggtgttg gtattattagacaaactaac aggacgctac ttagtggctt atattacaca 22500 tcactatcag gtgatttgttaggttttaaa aatgttagtg atggtgtcat ctactctgta 22560 acgccatgtg atgtaagcgcacaagcagct gttattgatg gtaccatagt tggggctatc 22620 acttccatta acagtgaactgttaggtcta acacattgga caacaacacc taatttttat 22680 tactactcta tatataattacacaaatgat aggactcgtg gcactgcaat tgacagtaat 22740 gatgttgatt gtgaacctgtcataacctat tctaacatag gtgtttgtaa aaatggtgct 22800 tttgttttta ttaacgtcacacattctgat ggagacgtgc aaccaattag cactggtaat 22860 gtcacgatac ctacaaactttaccatatcc gtgcaagtcg aatatattca ggtttacact 22920 acaccagtgt caatagactgttcaagatat gtttgtaatg gtaaccctag gtgtaacaaa 22980 ttgttaacac aatacgtttctgcatgtcaa actattgagc aagcacttgc aatgggtgcc 23040 agacttgaaa acatggaggttgattccatg ttgtttgttt ctgaaaatgc ccttaaattg 23100 gcatctgttg aagcattcaatagttcagaa actttagacc ctatttacaa agaatggcct 23160 aatataggtg gttcttggctagaaggtcta aaatacatac ttccgtccca taatagcaaa 23220 cgtaagtatc gttcagctatagaggacttg ctttttgata aggttgtaac atctggttta 23280 ggtacagttg atgaagattataaacgttgt acaggtggtt atgacatagc tgacttagta 23340 tgtgctcaat actataatggcatcatggtg ctacctggtg tggctaatgc tgacaaaatg 23400 actatgtaca cagcatcccttgcaggtggt ataacattag gtgcacttgg tggaggcgcc 23460 gtggctatac cttttgcagtagcagttcag gctagactta attatgttgc tctacaaact 23520 gatgtattga acaaaaaccagcagattctg gctagtgctt tcaatcaagc tattggtaac 23580 attacacagt catttggtaaggttaatgat gctatacatc aaacatcacg aggtcttgct 23640 actgttgcta aagcattggcaaaagtgcaa gatgttgtca acatacaagg gcaagcttta 23700 agccacctaa cagtacaattgcaaaataat ttccaagcca ttagtagttc tattagtgac 23760 atttataata ggcttgacgaattgagtgct gatgcacaag ttgacaggct gatcacagga 23820 agacttacag cacttaatgcatttgtgtct cagactctaa ccagacaagc ggaggttagg 23880 gctagtagac aacttgccaaagacaaggtt aatgaatgcg ttaggtctca gtctcagaga 23940 ttcggattct gtggtaatggtacacatttg ttttcactcg caaatgcagc accaaatggc 24000 atgattttct ttcacacagtgctattacca acggcttatg aaactgtgac tgcttggcca 24060 ggtatttgtg cttcagatggtgatcgcact tttggacttg tcgttaaaga tgtccagttg 24120 actttgtttc gtaatctagatgacaagttc tatttgaccc ccagaactat gtatcagcct 24180 agagttgcaa ctagttctgactttgttcaa attgaagggt gcgatgtgct gtttgttaat 24240 gcaactgtaa gtgatttgcctagtattata cctgattata ttgatattaa tcagactgtt 24300 caagacatat tagaaaattttagaccaaat tggactgtac ctgagttgac atttgacatt 24360 tttaacgcaa cctatttaaacctgactggt gaaattgatg acttagaatt taggtcagaa 24420 aagctacata acaccactgtagaacttgcc attctcattg acaacattaa caatacatta 24480 gtcaatcttg aatggctcaatagaattgaa acctatgtaa aatggccttg gtatgtgtgg 24540 ctactaatag gcttagtagtaatattttgc ataccattac tgctattttg ctgttgtagt 24600 acaggttgct gtggatgcataggttgttta ggaagttgtt gtcactctat atgtagtaga 24660 agacaatttg aaaattacgaaccaattgaa aaagtgcacg tccattaaat ttaaaatgtt 24720 aattctatca tctgctataatagcagttgt ttctgctaga gaattttgtt aaggatgatg 24780 aataaagtct ttaagaactaaacttacgag tcattacagg tcctgtatgg acattgtcaa 24840 atccatttac acatccgtagatgctgtact tgacgaactt gattgtgcat actttgctgt 24900 aactcttaaa gtagaatttaagactggtaa attacttgtg tgtataggtt ttggtgacac 24960 acttcttgct gctaaggataaagcatatgc taagcttggt ctctccatta ttgaagaagt 25020 caatagtcat atagttgtttaatatcatta aacacacaaa acccaaagca ttaagtgtta 25080 caaaacaatt aaagagagattatagaaaaa ctgtcattct aaattccatg cgaaaatgat 25140 tggtggactt tttcttagtactctgagttt tgtaattgtt agtaaccatt ctattgttaa 25200 taacacagca aatgtgcatcatatacaaca agaacgtgtt atagtacaac agcatcatgt 25260 tgttagtgct agaacacaaaactattaccc agagttcagc atcgctgtac tctttgtatc 25320 ttttctagct ttgtaccgtagtacaaactt taagacgtgt gtcggcatct taatgtttaa 25380 gattttatca atgacacttttaggacctat gcttatagca tatggttact acattgatgg 25440 cattgttaca acaactgtcttatctttaag atttgtctac ttagcatact tttggtatgt 25500 taatagtagg tttgaatttattttatacaa tacaacgaca ctcatgtttg tacatggcag 25560 agctgcaccg tttatgagaagttctcacag ctctatttat gtcacattgt atggtggcat 25620 aaattatatg tttgtgaatgacctcacgtt gcattttgta gaccctatgc ttgtaagcat 25680 agcaatacgt ggcttagctcatgctgatct aactgtagtt agagcagttg aacttctcaa 25740 tggtgatttt atttatgtattttcacagga gcccgtagtc ggtgtttaca atgcagcctt 25800 ttctcaggcg gttctaaacgaaattgactt aaaagaagaa gaagaagacc atacctatga 25860 cgtttcctag ggcattgactgtcatagatg acaatggaat ggtcattaac atcattttct 25920 ggttcctgtt gataattatattgatattac tttcaatagc attgctaaat ataattaagc 25980 tatgcatggt gtgttgcaatttaggaagga cagttattat tgttccagcg caacatgctt 26040 acgatgccta taagaattttatgcgaatta aagcatacaa ccccgatgga gcactccttg 26100 cttgaactaa acaaaatgaagattttgtta atattagcgt gtgtgattgc atgcgcatgt 26160 ggagaacgct attgtgctatgaaatccgat acagatttgt catgtcgcaa tagtacagcg 26220 tctgattgtg agtcatgcttcaacggaggc gatcttattt ggcatcttgc aaactggaac 26280 ttcagctggt ctataatattgatcgttttt ataactgtgc tacaatatgg aagacctcaa 26340 ttcagctggt tcgtgtatggcattaaaatg cttataatgt ggctattatg gcccgttgtt 26400 ttggctctta cgatttttaatgcatactcg gaataccaag tgtccagata tgtaatgttc 26460 ggctttagta ttgcaggtgcaattgttaca tttgtactct ggattatgta ttttgtaaga 26520 tccattcagt tgtacagaaggactaagtct tggtggtctt tcaaccctga aactaaagca 26580 attctttgcg ttagtgcattaggaagaagc tatgtgcttc ctctcgaagg tgtgccaact 26640 ggtgtcactc taactttgctttcagggaat ttgtacgctg aagggttcaa aattgcaggt 26700 ggtatgaaca tcgacaatttaccaaaatac gtaatggttg cattacctag caggactatt 26760 gtctacacac ttgttggcaagaagttgaaa gcaagtagtg cgactggatg ggcttactat 26820 gtaaaatcta aagctggtgattactcaaca gaggcaagaa ctgataattt gagtgagcaa 26880 gaaaaattat tacatatggtataactaaac ttctaaatgg ccaaccaggg acaacgtgtc 26940 agttggggag atgaatctaccaaaacacgt ggtcgttcca attcccgtgg tcggaagaat 27000 aataacatac ctctttcattcttcaacccc ataaccctcc aacaaggttc aaaattttgg 27060 aacttatgtc cgagagactttgtacccaaa ggaataggta acagggatca acagattggt 27120 tattggaata gacaaactcgctatcgcatg gtgaagggcc aacgtaaaga gcttcctgaa 27180 aggtggttct tctactacttaggtactgga cctcatgcag atgccaaatt taaagataaa 27240 ttagatggag ttgtctgggttgccaaggat ggtgccatga acaaaccaac cacgcttggt 27300 agtcgtggtg ctaataatgaatccaaagct ttgaaattcg atggtaaagt gccaggcgaa 27360 tttcaacttg aagttaatcaatcaagagac aattcaaggt cacgctctca atctagatct 27420 cggtctagaa atagatctcaatctagaggc aggcaacaat tcaataacaa gaaggatgac 27480 agtgtagaac aagctgttcttgccgcactt aaaaagttag gtgttgacac agaaaaacaa 27540 cagcaacgct ctcgttctaaatctaaagaa cgtagtaact ctaagacaag agatactaca 27600 cctaagaatg aaaacaaacacacctggaag agaactgcag gtaaaggtga tgtgacaaga 27660 ttttatggag ctagaagcagttcagccaat tttggtgaca ctgacctcgt tgccaatggg 27720 agcagtgcca agcattacccacaactggct gaatgtgttc catctgtgtc tagcattctg 27780 tttggaagct attggacttcaaaggaagat ggcgaccaga tagaagtcac gttcacacac 27840 aaataccact tgccaaaggatgatcctaag actggacaat tccttcagca gattaatgcc 27900 tatgctcgtc catcagaagtggcaaaagaa cagagaaaaa gaaaatctcg ttctaaatct 27960 gcagaaaggt cagagcaagatgtggtacct gatgcattaa tagaaaatta tacagatgtg 28020 tttgatgaca cacaggttgagataattgat gaggtaacga actaaacgag atgctcgtct 28080 tcctccatgc tgtatttattacagttttaa tcttactact aattggtaga ctccaattat 28140 tagaaagact attacttaatcactctttca atcttaaaac tgtcaatgac tttaatatct 28200 tatataggag tttagcagaaaccagattac taaaagtggt gcttcgagta atctttctag 28260 tcttactagg attttgctgctacagattgt tagtcacatt aatgtaaggc aacccgatgt 28320 ctaaaactgg tttttccgaggaattactgg tcatcgcgct gtctactctt gtacagaatg 28380 gtaagcacgt gtaataggaggtacaagcaa ccctattgca tattaggaag tttagatttg 28440 atttggcaat gctagatttagtaatttaga gaagtttaaa gatccgctac gacgagccaa 28500 caatggaaga gctaacgtctggatctagtg attgtttaaa atgtaaaatt gtttgaaaat 28560 tttccttttg atagtgatacaaaaaaaa 28588 <210> SEQ ID NO 2 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Porcine Transmissible Gastroenteritis V <400> SEQUENCE: 2cctaggattt aaatcctaag g 21 <210> SEQ ID NO 3 <211> LENGTH: 27 <212>TYPE: DNA <213> ORGANISM: Porcine Transmissible Gastroenteritis V <400>SEQUENCE: 3 gcggccgcgc cggcgaggcc tgtcgac 27 <210> SEQ ID NO 4 <211>LENGTH: 6 <212> TYPE: DNA <213> ORGANISM: Porcine TransmissibleGastroenteritis V <400> SEQUENCE: 4 gtcgac 6 <210> SEQ ID NO 5 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Porcine TransmissibleGastroenteritis V <300> PUBLICATION INFORMATION: <400> SEQUENCE: 5gctagcccag gcgcgcggta cc 22 <210> SEQ ID NO 6 <211> LENGTH: 11 <212>TYPE: DNA <213> ORGANISM: Porcine Transmissible Gastroenteritis V <400>SEQUENCE: 6 ctatggtata a 11 <210> SEQ ID NO 7 <211> LENGTH: 11 <212>TYPE: DNA <213> ORGANISM: Porcine Transmissible Gastroenteritis V <400>SEQUENCE: 7 aatgtaagtt a 11 <210> SEQ ID NO 8 <211> LENGTH: 11 <212>TYPE: DNA <213> ORGANISM: Porcine Transmissible Gastroenteritis V <400>SEQUENCE: 8 atttgcttga a 11 <210> SEQ ID NO 9 <211> LENGTH: 11 <212>TYPE: DNA <213> ORGANISM: Porcine Transmissible Gastroenteritis V <400>SEQUENCE: 9 ctatggtata a 11 <210> SEQ ID NO 10 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Porcine Transmissible Gastroenteritis V <400>SEQUENCE: 10 tttggtaaca cttcgttaac acacc 25 <210> SEQ ID NO 11 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Porcine TransmissibleGastroenteritis V <400> SEQUENCE: 11 ttacgagtca ttacaggtcc tgt 23 <210>SEQ ID NO 12 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: PorcineTransmissible Gastroenteritis V <400> SEQUENCE: 12 tttaagacgt gtgtcggcatctta 24 <210> SEQ ID NO 13 <211> LENGTH: 37 <212> TYPE: DNA <213>ORGANISM: Porcine Transmissible Gastroenteritis V <400> SEQUENCE: 13gaaattgact taaaagaaga agaagaagac catacct 37 <210> SEQ ID NO 14 <211>LENGTH: 10 <212> TYPE: DNA <213> ORGANISM: Porcine TransmissibleGastroenteritis V <400> SEQUENCE: 14 gtcgacgacc 10 <210> SEQ ID NO 15<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Porcine TransmissibleGastroenteritis V <400> SEQUENCE: 15 gaaatatttg tc 12

1. Method of preparing a DNA comprising steps, wherein (a) a DNAcomprising a full length copy of the genomic RNA (gRNA) of an RNA virus;or (b) a DNA comprising one or several fragments of a gRNA of an RNAvirus, which fragments code for an RNA dependent RNA polymetase and atleast one structural or nonstructural protein; or (c) a DNA having ahomology of at least 60 to the sequences of (a) or (b); is cloned into abacterial artificial chromosome (BAC).
 2. Method according to claim 1,wherein the DNA cloned into the BAC further comprises sequences codingfor several or all except one of the structural or non-structuralproteins of a virus.
 3. Method according to claim 1 or 2, wherein theDNA cloned into the BAC further comprises sequences encoding one orseveral heterologous gene.
 4. Method according to claim 3, wherein theheterologous gene encodes at least one antigen suitable for inducing animmune response against an infectious agent, at least one moleculeinterfering with the replication of an infectious agent, an antibodyproviding protection against an infectious agent, an immune modulator, acytokine, an immoenhancer or an anti-inflammatory compound.
 5. Methodaccording to one of claims 1 to 4, wherein the DNA cloned into the BAChas a size of at least 5 Kb.
 6. Method according to one of claims 1 to5, wherein the DNA cloned into the BAC has a size of at least 15 Kb. 7.Method according to one of claims 1 to 6, wherein the DNA cloned intothe BAC has a size of at least 25 Kb.
 8. Method according to one ofclaims 1 to 7, wherein the BAC comprises a sequence controlling thetranscription of the DNA cloned into the BAC.
 9. Method according to oneof claims 1 to 8, wherein one of the genes of the virus has beenmodified by substituting, deleting or adding nucleotides.
 10. Methodaccording to claim 9, wherein the gene controlling tropism of the virushas been modified.
 11. Method according to one of claims 1 to 10,wherein the gene controlling tropism of the virus has been substitutedwith the respective gene of another virus.
 12. Method according to oneof claims 1 to 11, wherein the DNA cloned into the BAC is capable ofbeing transcribed into RNA which RNA can be assembled to a virion. 13.Method according to of claim 12, wherein the virion is an infectious,attenuated, replication defective or inactivated virus.
 14. Methodaccording to one of claims 1 to 13, wherein the virus naturally has aplus strand genome.
 15. Method according to one of claims 1 to 14,wherein the virus is a picornavirus, flavivirus, togavirus, coronavirus,torovirus, arterivurs, calcivirus, rhabdovirus, paramixovirus,filovirus, bornavirus, orthomyxovirus, bunyavirus, arenavirus orreovirus.
 16. Method of preparing a viral RNA comprising steps, whereina DNA is prepared according to one of claims 1 to 15, the DNA isexpressed in a suitable host cell or the DNA is mixed with chemicals,biological reagents and/or cell extracts under conditions allowing thetranscription of the DNA and the viral RNA is isolated.
 17. Method ofpreparing a virion comprising steps, wherein a DNA is prepared accordingto one of claims 1 to 15, the DNA is expressed in a suitable host cellor the DNA is mixed with chemicals, biological reagents and/or cellextracts under conditions allowing the transcription and translation ofthe DNA and the virion is isolated.
 18. Method according to claim 17,wherein the virion is subsequently inactivated or killed.
 19. Method forpreparing a pharmaceutical composition comprising steps, wherein a DNAis prepared according to one of claims 1 to 15, a viral RNA is preparedaccording to claim 16 or a virion is prepared according to claim 17 or18 and is subsequently mixed with a pharmaceutically acceptable adjuvansor carrier.
 20. Method according to claim 19, wherein the pharmaceuticalis a vaccine for protecting humans or animals against an infectiousdisease.
 21. Method according to claim 19, wherein the pharmaceutical isused for gene therapy of humans or animals.
 22. DNA comprising sequencesderived from the genomic RNA (gRNA) of a coronavirus which sequenceshave a homology of at least 60% to the natural sequence of the virus andcode for an RNA dependent RNA polymerase and at least one structural ornonstructural protein, wherein a fragment of said DNA is capable ofbeing transcribed into RNA which RNA can be assembled a virion.
 23. DNAaccording to claim 22, further comprising a sequence encoding aheterologous gene.
 24. DNA according to claim 23, wherein theheterologous gene encodes at least one antigen suitable for inducing animmune response against an infectious agent, at least one moleculeinterfering with the replication of an infectious agent, an antibodyproviding protection against an infectious agent, an immune modulator, acytokine, an immonenhancer or an anti-inflammatory compound.
 25. DNAaccording to claim 22 or 24, wherein said fragment has a size of atleast 25 Kb.
 26. DNA according to one of claims 22 to 25, which furthercomprises sequences derived from a coronavirus coding for several or allexcept one of the structural or non-structural proteins of a virus. 27.DNA according to one of claims 22 to 26, which further comprisessequences derived from a coronavirus coding for all of the structural ornon-structural proteins of a coronavirus.
 28. DNA according to one ofclaims 22 to 27, further comprising a sequence controlling thetranscription of the viral gRNA.
 29. DNA according to one of claims 22to 28, wherein the sequence controlling transcription of the viral gRNAis the immediately early (IE) promoter of cytomegalovirus (CMV).
 30. DNAaccording to one of claims 22 to 29, wherein the sequence is flanked atthe 3′-end by a poly(A)tail, the ribozyme of the hepatitis 6 virus (HDV)and the termination and polyadenylation sequences of bovine growthhormone (BGH).
 31. DNA according to one of claims 22 to 30, wherein theviral sequences are derived from an isolate of the porcine transmissiblegastroenteritis virus (TGEV), murine hepatitits virus (MHV), infectiousbronchitis virus (IBV), bovine coronavirus (BoCV), canine coronavirus(CCoV), feline virus (FCoV), human coronavirus (HCoV), toroviruses orarterivurses.
 32. DNA according to one of claims 22 to 31, wherein thevirion is an infectious, non-infectious or replication deficient virus.33. DNA according to one of claims 22 to 32, wherein the sequence of thea structural or non-structural gene derived from the coronavirus hasbeen modified by substituting, deleting or adding one or severalnucleotides of the natural gene sequence.
 34. DNA according to one ofclaims 22 to 33, wherein the sequence of the S, N or M gene has beenmodified.
 35. DNA according to one of claims 22 to 34, wherein thesequence of the S gene derived from a coronavirus has been modified toobtain an attenuated virion.
 36. DNA according to one of claims 22 to35, wherein the sequence of the S gene derived from a coronavirus hasbeen modified to obtain a virion with a tropism differing from thetropism of the coronavirus.
 37. Vector comprising a nucleic acidaccording to one of claims 22 to
 36. 38. Vector according to claim 37,wherein the vector is a plasmid or bacterial artificial chromosome(BAC).
 39. Host cell comprising a nucleic acid according to one ofclaims 22 to
 38. 40. E. coli deposited under CECT 5265 at the SpanishCollection of Type Cultures.
 41. Method for producing a recombinantvirion or a recombinant viral RNA comprising steps, wherein a DNAaccording to one of claims 22 to 38 is introduced into a host cell, hostcells containing the DNA are cultivated under conditions allowing theexpression thereof and the recombinant virion or viral RNA is recovered.42. Method for producing a recombinant virion or a recombinant viralRNA, wherein a DNA according to one of claims 22 to 38 is mixed withchemicals, biological reagents and/or cell extracts under conditionsallowing the transcription of the DNA and the recombinant virion orviral RNA is recovered.
 43. Method according to claim 41 or 42, whereinthe DNA is a DNA according to one of claims 23 to
 38. 44. Virionobtainable by a method according to one of claims 41 to
 43. 45. Virionaccording to claim 44, wherein the virion is an infectious, attenuated,replication defective or inactivated virus.
 46. Virion according toclaim 44 or 45, wherein the virion comprises a modified S, M or N gene.47. Virion according to claim 46, wherein modification of the S genegives raise to an attenuated virus.
 48. Virion according to claim 46,wherein modification of the S gene gives raise to a virion with alteredtropism.
 49. Viral RNA obtainable by a method according to claim 41 to43.
 50. Pharmaceutical preparation comprising a nucleic acid accordingto one of claims 22 to 38, a host cell according to claim 39 or 40, avirion according to one of claims 41 to 46 or a viral RNA according toclaim
 49. 51. Vaccine capable of protecting an animal or a human againstdeseases caused by an infectious agent comprising a nucleic acidaccording to one of claims 22 to 38, a host cell according to claim 39or 40, a virion according to one of claims 41 to 48 or a viral RNAaccording to claim
 49. 52. Vaccine according to claim Si, wherein thenucleic acid comprises sequences encoding least one antigen suitable forinducing an immune response against the infectious agent, at least onegene interfering with the replication of the infectious agent or anantibody providing protection against said infectious agent,
 53. Vaccineaccording to claim 51 or 52, wherein said virion vector expresses atleast one replication interfering molecule, an antigen capable ofinducing a systemic immune response and/or an immune response in mucousmembranes against different infectious agents that propagate inrespiratory or intestinal mucous membranes or in other tissues. 54.Multivalent vaccine capable of protecting an animal or a human againstthe infection caused by more than one infectious agent, that comprisesmore than one nucleic acid according to one of claims 22 to 38, a hostcell according to claim 39 or 40, a virion according to one of claims 41to 48 or a viral RNA according to claim 49, each of which expresses anantigen adequate for inducing an immune response against each of saidinfectious agents, an interfering molecule or an antibody providingprotection against each of infectious agents.
 55. Vaccine according toone of claims 51 to 54 further comprising a pharmaceutically acceptablecarrier or diluent.
 56. Method of preparing a DNA according to one ofclaims 22 to 38 comprising steps, wherein an interfering defectivegenome derived from a coronavirus is cloned under the expression of apromotor into a BAC vector and the deleted sequences within thedefective genome are re-inserted.
 57. Method of preparing a DNAaccording to claim 56, wherein toxic sequences within the viral genomeare identified before re-insertion into the DNA.
 58. Method of preparinga DNA according to claim 56 or 57, wherein the toxic sequences withinthe viral genome are the last sequences to be re-inserted whencompleting the genome.
 59. An infective clone derived from a coronavirusthat comprises a full-length copy of complementary DNA (cDNA) to thegenomic RNA (gRNA) of a coronavirus, cloned under atranscription-regulatory sequence.
 60. Infective clone according toclaim 59, in which said coronavirus is an isolate of the porcinetransmissible gastroenteritis virus (TGEV).
 61. Infective cloneaccording to claim 59 or 60, in which said promoter is the immediatelyearly (IB) promoter of expression of cytomegalovirus (CKV). 62.Infective clone according to one of claims 59 to 61, wherein saidfull-length cDNA is flanked at the 3′-end by a poly(A) tail, theribozyme of the hepatitis delta virus (HDV), and the termination andpolyadenylation sequences of bovine growth hormone (BGH).
 63. Infectiveclone according to one of claim 59 to 62, wherein said infective cDNA iscloned in a bacterial artificial chromosome (BAC).
 64. A procedure forobtaining of an infective clone according to any of claims 59 to 63,which comprises constructing the full-length cDNA from the gRNA of acoronavirus and to assemble the transcription-regulatory elements. 65.Procedure according to claim 64, in which the construction of thefull-length cDNA of the gRNA of a coronavirus comprises: (i) cloning aninterfering defective genome derived from said coronavirus under apromoter of expression in a BAC; (ii) completing the deletions of saidinterfering defective genome and regenerating the deleted sequences withrespect to the infective gRNA; (iii) identifying the toxic sequences forthe bacteria in which it is going to be cloned, removing the toxicsequences, and inserting said toxic sequences just before effecting thetransfection in eukaryotic cells to obtain the cDNA clone, correspondingto the gRNA of the coronavirus.
 66. A recombinant viral vector thatcomprises an infective clone according to any of claims 59 to 63, orobtainable according to the procedure of either of claims 58 or 59,modified to contain a heterologous nucleic acid inserted into saidinfective clone under conditions that allow said heterologous nucleicacid to be expressed.
 67. Vector according to claim 60, in which saidheterologous nucleic acid is selected between a gene and a gene fragmentthat codes a gene product of interest.
 68. A method for producing aproduct of interest that comprises cultivating a host cell that containsa viral vector according to either of claims 60 or 61 under conditionsthat allow the heterologous nucleic acid to be expressed and the productof interest to be recovered.
 69. A method for producing a modifiedrecombinant coronavirus that contains a heterologous nucleic acid in asequence of cDNA corresponding to the genome of a coronavirus, whichcomprises introducing a viral vector according to either of claims 60 or61 into a host cell, cultivating said host cell containing said viralvector under conditions that allow the viral vector to be expressed andreplicated, and the virions obtained from the modified recombinantcoronavirus to be recovered.
 70. A vaccine capable of protecting ananimal against the infection caused by an infectious agent thatcomprises (i) at least one viral vector according to claim 60 or 61 thatexpresses at least one antigen suitable for inducing an immune responseagainst said infectious agent, or an antibody that provides protectionagainst said infectious agent, along with, optionally, (ii) apharmaceutically acceptable excipient.
 71. Vaccine according to claim70, in which said viral vector expresses at least one antigen capable ofinducing a systemic immune response and/or an immune response in mucousmembranes against different infectious agents that propagate inrespiratory or intestinal mucous membranes.
 72. A multivalent vaccinecapable of protecting an animal against the infection caused by morethan one infectious agent, that comprises (i) a viral vector accordingto claim 60 or 61, that expresses an antigen adequate for inducing animmune response against said infectious agents, or antibodies thatprovide protection against said infectious agents, along with,optionally, (ii) a pharmaceutically acceptable excipient.
 73. Amultivalent vaccine capable of protecting an animal against theinfection caused by more than one infectious agent, which comprises (i)more than one viral vector according to claim 60 or 61, each one ofwhich expresses an antigen adequate for inducing an immune responseagainst each one of said infectious agents, or antibodies that provideprotection against each one of said infectious agents, along with,optionally, (ii) a pharmaceutically acceptable excipient.
 74. A methodfor producing a recombinant coronavirus that comprises introducing aninfective clone according to any of claims 59 to 63, or obtainableaccording to the procedure of either of claims 64 or 65 into a hostcell, cultivating said host cell that contains the infective clone underconditions that allow the infective clone to be expressed andreplicated, and recovering virions obtained from the recombinantcoronavirus containing the complete genome of the coronavirus.